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CONTENTS

Being reprints of studies issued in 1910.

Sie IN PCUENCE OF ALCOHOL AND OPTHER ANAESTHET-

ICS ON EMBRYONIC DEVELOPMENT
By Charles R. Stockard.

Am, Jour. of Anat., Vol X, 369-392 and 20 text-figures.

’

) THE INDEPENDENT ORIGIN AND DEVELOPMENT OF THE

GERYSETALLINE LENS By Charles R. Stockard.

Am. Jour. of Anat., Vol. X, 393-423, 28 text-figures and 2 plates.

PaaS Or TISSUE “GROWTH. Ll THE RATE OF RE-

GENERATIVE GROWTH IN DIFFERENT SALT SOLU-
TIONS By Charles R. Stockard.
Arch. t. Entw.-Mech., Vol. XXIX, 15-24, and 4 text-figures.

Souths OF TISSUE GROWTH. IV. THE INFLUENCE OF

REGENERATING TISSUE ON THE ANIMAL BODY
By Charles R. Stockard.
Arch. f. Entw.-Mech., Vol. XXIX, 24-32.

ihe EXPERIMENTAL PRODUCTION OF VARIOUS EYE

ABNORMALITIES AND AN-*ANALYSIS OF THE DE-
VELOPMENT OF THE PRIMARY PARTS OF THE EYE
By Charles R. Stockard.

Arch. f. vergleichende Ophthalmologie, Bd. I, 473-480, and 2 text-figures.

) THe ANATOMY OF THE THYROID GLAND OF ELASMO-

BRANCHS, WITH REMARKS UPON THE HYPOBRAN-
CHIAL CIRCULATION IN THESE FISHES
By J. S. Ferguson.

Amer. Jour. of Anat., Vol. XI, 151-210, and 20 text-figures.

ft PHYROID GLAND OF THE TELEOSTS

By J. F. Gudernatsch.
Jour. of Morphology, Vol. XXI, No. 4 Suppl., 709-783, 21 text-figures and 5 plates.

ON THE EFFECT OF EXTERNAL CONDITIONS ON THE
REPRODUCTION OF DAPHNIA By J. F. McClendon
Amer. Naturalist, Vol. XLIV, 404-411.

StHeE DEVELOPMENT OF ISOLATED BLASTOMERES OF

THE FROG’S EGG By J. F. McClendon.

Amer. Jour. of Anat., Vol. X, 425-430 and 2 text-figures.

10) ON THE EPFECT OF CENTRIFUGAL FORCE ON THE

FROG’S EGG By J. F. McClendon.
Arch. f. Zellforschung, Bd. V., 385-393, and 9 text-figures.

11. FURTHER STUDIES ON THE GAMETOGENESIS OF PAN-
DARUS SINUATUS, SAY By J. F. McClendon.
Arch. f. Zellforschung, Bd. V, 229-234, 1 text-figure and Pl. XVII.
12° ON THE DYNAMICS OFVCEEL DIVISION] Thea niece
TRIC CHARGE ON COLLOIDS IN LIVING CELESa
“THE ROOT TIPS3@ERSELANTS By J. F. McClendon.
Arch. f. Entw.-Mech., XXXI, 80-90, 2 text-figures and Pl. III. :

13. ON THE DYNAMICS OF CELL DIVISION. 11 CHANGES
PERMEABILITY OF DEVELOPING EGGS TO ELEC
TROLYTES By J. F. McClendon.

Amer. Jour. of Physiology, Vol. XXVII, 240-275.

14. ON ADAPTATIONS IN STRUCTURE AND HABITS OF SOME
MARINE ANIMALS OF TORTUGAS, FLORIDA
By J. F. McClendon. ~

Publication of the Carnegie Institution No. 132, 55-62, 1 text-figure and 2 plates.

THE INFLUENCE OF ALCOHOL AND OTHER ANAS-
THETICS ON EMBRYONIC DEVELOPMENT

CHARLES R. STOCKARD

Anatomical Laboratory, Cornell University Medical School, New York City,

WITH TWENTY TEXT FIGURES

The adult nervous system is peculiarly sensitive in its responses
to the influence of alcohol and other anesthetics. The writer has
found it to be equally true that alcohol and anesthetics exert a
most striking influence over the development of the central ner-
vous system and the organs of special sense. There is consider-
able variation in the way in which the several anesthetics act on
the developing animal; some of them, such as ether and chlore-
tone, producing effects of a general nature, while alcohol and mag-
nesium are more localized or specific in their action. A similar
statement is true for the actions of different anesthetics on the
adult body. .

In attempting to explain the occurrence of asymmetrically
monophthalmic, cyclopean and blind individuals among fish
that had been developed in solutions containing magnesium, the
writer advanced the hypothesis that the anesthetic property of
Mg was the causal factor. Many reasons for such a view were put
forward in a paper on the artificial production of these monsters
(1909). To experimentally test this hypothesis various other
anesthetic agents have been used and all of them to a higher or
lower degree inhibit the development of the optic vesicles in fish
embryos, and thus give rise to various ophthalmic defects. Alco-
hol is most decided in its action, causing in some experiments as
high as 90 to 98 per cent of abnormal eyes, generally cyclopean,
which far surpasses the highest results obtained with Mg.

The effect of alcohol on the general development of the nervous
system is more pronounced than that of Mg, and only a few of the

THE AMERICAN JOURNAL OF ANATOMY, VOL. 10 No. 3.

370 Charles R. Stockard.

alcoholic specimens ever develop sufficiently to hatch and swim
about as de the Mg embryos. An explanation for this may
be that Mg exerts an influence to inhibit dynamic processes, such
as the out-pushing of the optic vesicles, while alcohol acts more
especially on the nervous tissues. Mayer (1908) has shown that
Mg inhibits muscular contractility without affecting in any way
the nervous impulse or nervous rhythm.

The eye defects, it must be remembered, have only been ob-
tained in solutions of one or another anesthetic; the many other
salt and sugar solutions which have been experimented with dur-
ing four years (06 and ’07) have failed entirely to produce similar
results.

The most important outcome of these experiments has been to
prove conclusively that many monsters which occur in nature
may be artificially produced by changing the environment of the
normally developing eggs. The present experiments will demon-
strate that this may be done even after development has pro-
ceeded for some time. These anomalous structures being the
results of external influences and not germinal variations are to
some extent within scientific control. A promising field is thus
opened in the devising of means to control or regulate the devel-
opment of the embryo and possibly to obviate certain monstrous
conditions at least. Such possibilities were of course beyond our
reach if defective germ cells were actually the cause of these mon-
sters.

Mall (’08) has brought forward evidence to show that im-
proper placentation or unfavorable developmental environment
is responsible for most human monstrosities, many of which are
aborted before reaching term. There is evidently much need of
investigation aiming toward the control and regulation of the
. developmental environment of mammals.

METHOD AND MATERIAL

The eggs of the fish, Fundulus heteroclitus, were used in all of
the experiments. The method of treatment varies somewhat
for the different solutions employed, so that it is best to describe
each separately.

Effect of Aleohol and Anzesthetics. ayal

Alcohol solutions were prepared in sea-water on the percentage
basis. The strength used being 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,
18 and 20 per cent, 60 cc. of each solution was poured into finger
bowls and from 60 to 100 eggs, in the early cleavage stages, four
and eight cells, were placed in each bowl. The stronger solu-
tions killed all of the eggs, and those from 3 to 9 per cent gave the
best results. In the 3 per cent alcohol solutions at times as many
as 90 in every 100 embryos showed abnormal conditions of the
eyes, being either eyeless, asymetrically monophthalmic or cyclo-
pean, while in one experiment in a 5 per cent solution of alcohol
in sea-water, there were 146 ophthalmic monsters against only 3
individuals with two separate eyes.

Chloretone of 0.1 per cent and 0.066 per cent in sea-water
caused abnormalities similar to those produced by other anes-
thetics. This substance is more general in its anesthetic action
than either alcohol or Mg, as will be seen in the discussion to fol-
low.

Ether and chloroform also produce rather general effects on the
developing embryo, yet a small percentage of cyclopean monsters
occur among the embryos which are treated with 60 cc. of sea-
water to which 2,2.5, and 3 cc. of ether has been added. In solu-
tions of chloroform of about the same proportions and slightly
weaker, a few monsters occurred of the type common to the other
anesthetics. Chloroform is rather toxic in its action on these
eges, large numbers dying in the weaker solutions, while others
are so inhibited in their development, that various abnormal con-
ditions follow.

Eggs were not exposed to any of the above anesthetics for
more than twenty-four or thirty-six hours. They were then
placed in pure sea-water and continued development showing
the abnormal conditions of the eyes and central nervous system
that had been induced by their sojourn in the unusual environ-
ment.

Similar Mg solutions to those formerly employed were again
used. A gram-molecular solution of MgCl, in distilled water was
titrated and kept, to be diluted with sea-water to the proper
strength just before the eggs were placed in it. The most

ate Charles R. Stockard.

favorable results were obtained in solutions of 16, 17, 18, 19, 20,
21 and 22 ec: of molecular MgCl, made up to 60 ec. by the addition
of a sufficient amount of pure sea-water; e.g., 44 cc. of sea-water
was added to the 16 cc. of molecular MgCl, and 43 cc. of sea-water
to the 17 cc. of MgCl. The solutions are, therefore, 16) 17 18
ete., parts MgCl. to sea-water. In the ¢» solution 66 per cent
of the embryos were cyclopean in one of the experiments. Eggs
were exposed to the action of Mg shortly after fertilization and at
various other times until they reached an early periblast stage, or
were fourteen hours old, all with similar results. Although the ~
most favorable time for introducing the eggs into Mg solutions
is during the eight-celled stage. The developing embryos were
returned to pure sea-water after the third day. The Mg is so
slightly toxic that eggs may be kept in it and will continue to
develop; the embryos actually hatch and swim about in the solu-
tion, being, however, slightly slower in their developmental rate
and not so hardy as the specimens which are returned to the sea-
water.

Tue Action oF ALCOHOL ON DEVELOPMENT

Weak solutions of alcohol exert a most decided effect on the
developing fish embryos, causing deformities of the central nervous
system, the eyes, and ears in a very large percentage of the speci-
mens.

a. Defects of the eyes

Typical cases of cyclopia showing in the different specimens
all gradations, from merely closely approximated eyes, hour-glass
eyes with two pupils and two lenses, oval eyes having the two
component intimately associated, typical median cyclopean eyes
with scarcely an indication of their double nature and extremely
small ill-formed cyclopean eyes, were present in the weak alcohol
solutions. All of these have been fully described in a former
paper (09) on the artificial production of cyclopea as a result of
the action of Mg. The alcohol monsters in some cases also
present various degrees of the monophalmicum asymmetricum

Effect of Aleohol and Anesthetics on Development. 373

defect which was common in the Mg experiments. Individuals
may have one normal eye and the other eye in different conditions
of arrested development from slightly small and defective to
entirely absent, see figs. 1 and 2. An important point that was
brought out by the alcohol monsters which was not noticed in
the magnesium specimens is the fact that some of the embryos
have both eyes equally small and defective, figs. 3, 8, 9, 10, and 11.
The two eyes are symmetrically defective and the head appears
to have small eye-spots instead of normal eyes; compare figs. 3,
and 7, 9, 10 and 11 and 12. Finally, as in Mg solutions so also
in the alcohol, many eyeless individuals are present .

The eye in some of the alcohol monsters is rather different from
that found in the Mg embryos, and may possibly serve to indicate
something of the condition of the eye anlagen in the brain. Many
embryos possess optic cups with their concave surfaces facing
almost directly toward the median sagittal plane of the head. In
life the eye presents a heavily pigmented solid convex surface to
the side of the head instead of the usual open pupil through which
the lens may be seen within the cavity of the optic cup. Fig.
5 and 6 show front and lateral views of such an embryo; the side
view indicates the peculiarly solid convex object seen when looking
towards the lateral eye. Fig. 13 represents a section through
thiseye. The choroid coat of the eye-ball is pressed close against
the body wall on the sides of the head and the concave retinal
surfaces which should face outward are turned directly towards
one another; a lens lies between the two eye components which are
really separate except along their dorsal borders. Fig. 4 shows
a somewhat similar specimen in which the eyes are entirely sepa-
rate yet they have an arrangement almost identical to that
just described. The eyes face the mid-plane of the head and
turn their convex choroid coats out against the body wall. The
only place at which the inner face of the eyes touches the ecto-
derm is the ventral body wall and from this a lens has arisen and
lies between the two eyes. Fig. 14 illustrates a section through
these eyes, in which a most peculiar arrangement exists. Optic
fibers probably arising from the ganglionic layer of the retina,
(although this can not be positively demonstrated in the sections),

374 Charles R. Stockard.

Camera DRAWINGS OF Livine FunpuLUS EMBRYOS

Fic. 1. An embryo twenty days old which was treated with5 per cent alcohol.
Only one eye is formed and it faces in a ventro-median direction, instead of to-
wards the lateral wall of the head.

Fic. 2. An asymmetricum nonophthalmicum monster with one almost typical
eye while the other eye is small and poorly formed, from a 4 per cent solution of
alcohol in sea-water.

Fra. 3. An embryo with both eyes small and closely approximated; the eyes
face ventrally. This type is common in all of the anesthetic solutions.

Fie. 4. A nineteen day embryo from 5 per cent alcohol. The two eyes are not
connected yet their convex surfaces are turned out against the lateral walls of the
head and the pupils face the median plane. A single lens hes between the two
eyes. Fig. 14 shows a section of these eyes.

Fieg.5. Anineteen day embryo from 5 per cent alcohol. This front view shows
the two eyes joined dorsally and facing one another with the lens between them. -

Fig. 6. shows a lateral view of the same head with the convex choroid surface
of the eye-ball close against the side of the head. Fig. 18 illustrates a section
through these eyes.

Fig.7. A normal Fundulus embryo when eight days old drawn to the same scale
as the monsters.

Fic. 8. A twenty day embryo from 5 per cent alcohol; the eyes are small and
defective as shown in the section fig. 10.

Ke)

Effect of Aleohol and Anesthetics.

376 Charles R. Stockard.

are collected into optic nerves which pursue extraordinary courses;
instead of passing through the outer retinal layers and the choroid
coat they take an almost directly opposite course and run across
what should be the optic cup cavity (the humor cavity in these
specimens is filled with loose cellular tissue) and out through the
wide-open ‘‘pupil,”’ forming a perfect cross and then passing into
the base of the brain to end in the optic lobes.

The position of the lens must not be supposed to determine the
actual pupil region of these eyes, as the lens clearly lies between
them; see fig. 4 of the living embryo. The wide open pupils of
the two eyes face or lead directly into one another. The position
might be taken that the entire arrangement represents. one
large eye; this is not true however since the eyes are entirely
separate in all of the sections and the optic cross could scarcely be
expected to exist within the base of the eye itself as would be the
case if this were one huge eye. The eyes really hang down from
the brain as two large retinal disks. Fie. 11 represents a similar
case with the eyes rather more flattened out laterally, and the
existence of all gradations between the two conditions substan-
tiates the above statement.

The retinal layers of these eyes which are nineteen days old,
are poorly differentiated, the inner layer consisting merely of
indefinitely arranged cells; a better differentiation is usually
attained in normal specimens by the sixth or seventh day. A
comparison of Figs. 13 and 14 with Fig. 12 illustrates in a way the
more definite orientation of the inner retinal cells in the normal
individual when compared with the defective eyes. It will also
be noticed that the lenses in the two-eyed specimen are surrounded
by clear humor spaces while the lens in the defective eyes lies
buried in loosely arranged cellular tissue.

The right retina of the monster faces in the same direction as
the left retina of the ordinary individual yet there is no indication
ofa reversal of the layers which might possibly beimagined in such
a case.

The optic stalk was scarcely formed in these eyes, which is
not infrequently true in cyclopia. The path of. the optic
nerve is, therefore, evidently not that usually taken along the

Effect of Alcohol and Anesthetics on Development. 377

Fic. 9. Asection through the small defective eyes of an eight day embryo, after

treatment with a4 per cent solution of etherinsea-water. The brainis narrow and

poorly developed.
Fig. 10. A section through the head of the embryo shown in fig. 8. Both of the

eyes are small and defective with ill-fitting lenses and face in a ventral direction.
Fie. 11. A section through the eyes of a monster commonly found in the alco-

hol solutions. The eyes are joined beneath the bilateral brain and face ventrally.
Fig. 12. A section through the eyes of a normal thirteen day embryo.

378 Charles R. Stockard.

optic stalk, but the optic nerve fibers grow directly into the brain
from the region of the eye cavity itself.

It is interesting to find in this connection that Lewis (70)
describes in his experiments on tadpoles a strikingly similar
course pursued by the nerves arising from some of the optic cups
he had transplanted to various positions along the hind brain
region of the embryos. He states that :

In a few of these somewhat irregular transplanted eyes the optic nerve
takes a very curious course, passing across the cup cavity from the gang-
lionic layer, through the pupil and then into the mesenchyme, ending
there. In both of these experiments a small bundle of optic nerve fibers
pierces the retina as faras the pigment layer. In transplanting these
eyes the ganglionic layer was probably injured in such a way as to inter-
fere with the normal path of the nerve fibers, and so they have.prob-
ably followed the path of least resistance through the pupil and out into
the mesenchyme.

Fic. 13. A section of the embryo shown in figs. 5 and 6. The optic cups are
joined dorsally and face the median plane of the head; a lens lies between them
and is surrounded by loose cellular tissue instead of by the humor,

Fic. 14. Section of the eyes in the embryo shown in fig. 4. The optic cups are
not joined yet they face towards one another with their convex choroid surfaces
pressed close against the head wall. The optic nerves run across what should be
the humor chamber of the eyes and out through the wide pupils to form a perfect
cross; the optic fibers then enter the brain floor. A lens lies between the eyes.

Effect of Alcohol and Ansesthetics. 379

Lewis’ illustrations are similar to Fig. 14 in so far as the course
of the optic nerve is concerned. These experiments would seem
to indicate that the direction taken by the optic nerve fibers is
not firmly fixed but that they may pursue an almost reverse
direction from that generally followed. Many of the eyes in the
writer’s specimens show various conditions of this kind and he
must agree with Lewis in the conclusion that

It would seem to me impossible to explain these various conditions
of the optic nerve on any other basis than that they are outgrowths of
nerve cells of the ganglionic layer of the retina.

Direct evidence is thus furnished for the outgrowth theory of the
nerve fiber which has been so ably supported in the last few years
by Harrison’s (’08) experiments.

The present experiments warrant the following explanation
for incomplete cyclopean eyes, or double eyes, when compared with
the usual condition.

In normal development the eye anlagen push out from the ven-
tro-lateral borders of the brain and turn dorsally as indicated in
the diagram, fig. 15 A. The abnormal individuals with two
eyes facing the median plane also have them more ventrally
situated in relation to the brain, and it may be supposed that when
the eyes arose from the brain their formation was directed ven-
trally instead of dorsally, fig. 15 B. This causes the eyes to hang
below the brain and face one another as already shown in figs.
4, 5, 6, 13, and 14, instead of turning dorsally and facing outward
as in figs, 11 and 12.

Similar conditions are also found in the development of a single
eye. Fig. 1 shows an embryo with an eye on the right side only,
yet this eye faces the median plane and is unusually ventral in
position; it probably arose as indicated in the diagram fig. 15 D,
where as other single-eyed individuals, the commoner type, have
an eye looking out from the usual lateral position, fig. 15 C.

From these conditions we may determine whether cyclopia is
brought about by a failure of certain central tissues of the brain
to develop, thus allowing the eye anlagen to come together as
Lewis (’09) has suggested, or whether through a lack of develop-
mental energy necessary for the optic cups to grow dorsally and

380 Charles R. Stockard.

outward to meet the ectoderm as the writer (’09) has supposed.
Considering the case of the single eye it might be held that the fail-
ure of certain central tissues of the brain to develop would cause
the eye to arise too near the median line, but this lack of central
tissue does not explain why the eye faces the median plane instead
of the lateral head wall, and it is much less able to account for the
absence of the eye on the opposite side of the head. On the other
hand, if the conditions are due to a lack of the necessary develop-
mental energy or an anesthesia produced by the experiment, then
itis evident that although one eye does succeed in pushing out from
the brainit might not have sufficient developmental energy to grow
dorsally and outward to the lateral body wall, but droops, as
it were, into a more ventro-median position and faces in toward
the median plane. Thus one-half of an incomplete cyclopean
eye is formed. The other eye was entirely suppressed, lacking
the energy necessary to push itself out from the brain. This
inequality in the developmental powers of the two eyes is indi-
cated by their frequent asymmetrical condition.

The two eye components do not always face the median plane
and in such cases the eyes merely fail to grow out laterally. They
come off ventrally from the brain and either face in a ventral
direction or grow so as to face outward.

The experiments fail to give any definite clue as to where the
optic anlagen are located in the brain before they become visible,
although Lewis’ operations on the embryoni¢ shields of older
embryos would seem to indicate that at that time the anlagen
occupied somewhat lateral positions.

It is clear from the foregoing consideration that alcohol has
the power to induce the same typical ophthalmic defects that were
formerly described in the embryos from the Mg solutions. The
property common to both Mg and alcohol is their anesthetic
effect on animals. The writer concludes that cyclopia, mon-
ophthalmicum asymmetricum and entire absence of eyes, all of
which are more or less arrested or inhibited condition of develop-
ment, result from anesthesia during certain embryonic stages.
Of course this may not be the sole cause of such defects; on the
contrary the fact that they are produced in this way would indicate

Effect of Aleohol and Anesthetics on Development. 381

Fig. 15. A diagram illustrating several positions taken by the optic cups in
development. A, the usual case in which both cups push out trom the brain and
turn dorsally so as to face the lateral head wall with their convex choroid surfaces
towards the brain; see fig. 12. B, the optic cups push out from the brain but in-
stead of growing dorsally, they hang ventrally and face in towards the median
plane, with their convex choroid surfaces against the outer head wall; see figs. 18
and 14. C and D, the same conditions are often realized when only one optic ves-
icle arises from the brain.

382 Charles R. Stockard.

that any factor which might come in during early development
to lower the developmental energy could possibly induce similar
defects. In mammals such monsters probably arise as the result
of some weakening or debilitating influence of the environment
during early developmental stages, which need only have acted
for a short space of time.

b. Defects of the auditory organs

A very pronounced suppression in the development of the audi-
tory apparatus is often noticed in the embryos which have been
treated with weak solutions of alcohol. In many individuals only
one ear exists. When this condition is found in an embryo with
only one eye, two unequally developed eyes or a cyclopean eye
with asymmetrical components, it is of interest to find that in all
-ases observed, the ear is on the same side of the head as the better
formed eye. In rare cases both ears are absent, and again it
often happens that the ears are ‘apparently normal while the
eyes are deformed. Fig. 16, a horizontal section through the
head, shows two small abnormal eyes with a lens between them
and two perfect ears with cartilaginous capsules, near the hind
brain. Fig. 18, which is a section through the ear region of the
embryo shown in fig. 4, illustrates two poorly formed ears; on the
right side the ear issmall and two semicircular canals are represented
only by their ampulle, the epithelial lining of which forms papille
of cells with long hair-like processes growing from them as is
indicated in the drawing. The left ear is almost entirely absent,
its median section showing only the small cavity and ampullary
papilla seen in the figure. Both ears, however, are surrounded by
well formed cartilaginous capsules.

A remarkably abnormal ear is seen in fig. 17. The auditory
vesicles have united so as to occupy a dorsal position above the
posteriorend of the brain. Only two semicircular canalsare devel-
oped on each side. The cartilaginous capsules in this case seem
unable to meet the situation and extend for only a portion of the
way around the huge auditory cavity. This union of the lateral
auditory vesicles, although formed by an entirely different princi-
ple, suggests the large double cyclopean eye.

383 Effect of Aleohol and Anesthetics.

The final persistence of the ampulla-like cavities seems to be
the rule, these structures being present even when all other por-
tions of the internal ear are absent. The ampulle of the canals
are perhaps particularly useful as organs of equilibration in these
animals, and their stuborn persistence may be indicative of an
ancient origin and suggests the primary function of the ear as an
organ of equilibrium.

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Fig. 16. A horizontal section through the head of a thirteen day embryo from
a 5 per cent alcohol solution. Two very small and defective eyes have alens be-
tween them, while more posteriorly two perfectly formed auditory vesicles areseen
with cartilaginous capsules surrounding them.

Fig. 17. A section through the auditory region of an eight day embryo treated
with a4 per cent ether solution. The two auditory vesicles unite dorsally to form
a huge cavity above the medulla and spinal cord. This embryo also shows an in-
complete cyclopean eye and spina-befida. se, semicircular canal; a, auditory
vesicle, ; ph, pharynx. z

Fig. 18. A section through the auditory vesicle of the embryo shown in fig. 4
and section fig. 14. The entire auditory vesicle is suppressed except the ampulle
of the semicircular canals. The right ampulla is larger and shows a papilla with
hair-like fibers, while the left ampulla is almost completely closed, yet, it too, shows
the papilla with projecting hairs. The cartilaginous capsules are small and thus
adjusted to the tiny ear parts.

384 Charles R. Stockard.

Although all parts of the ear are absent the cartilaginous cap-
sules are present. The shape and size of the capsules, however,
seem to be adjusted to that of the auditory vesicle when any part
of it exists, as is indicated on the two sides of fig. 18.

c. Defects of the central nervous system

The abnormalities of the brain shown by the specimens treated
with alcohol might easily form the subject of an extensive mono-
graph so various and numerous are they. Only a few of them
will be briefly mentioned.

In rare cases the brain is almost normal; the fore brain, however,
is usually very narrow and gives to the head a characteristically
pointed appearance. Dorsal hernize at times occur in the region
of the optic lobes and the hind brain. The histological structure
of the brain is often peculiarly abnormal in both the arrangement
and the appearance of the cells. The cells may be hyaline and in
the region of the central cavity fail to take the stains. They may
even be diffusely scattered in peculiarly defective specimens.

The spinal cord in some individuals also shows the hyaline
appearance about its central canal, and spina-bifida in not uncom-
mon. The latter condition no doubt results from the general
inhibition in rate of development which is constantly true for
the specimens in the alcohol solutions. The germ ring is slow in
surrounding the yolk and consequently the trunk region of the
early embryo is abbreviated. This condition interferes with the
median cell proliferation forming the spinal cord so that a split
or divided cord results and may extend for various distances in
the trunk region. Fig. 19 shows a section through a trunk region
with a double cord, ch, the notocord, nch, is also divided. Fig.
20 is a more posterior section of the same embryo and shows the
cord and notocord again single as they are in more anterior region
than that shown by fig. 19.

Many of the defects of the central nervous system are of a
general nature and almost any substance that inhibits or inter-
feres with the normal developmental rate may cause them. The
writer does not intend to conyey the idea that these are characteris-

Effect cf Alcohol and Anesthetics on Development. 385

Fie. 19. A section through the trunk region of an eight day embryo from a 4
per cent ethersolution. Thespinal cord, ch, isdouble (spina-bifida) and the noto-
chord, nch, is divided into three parts.

Fig. 20. A more posterior section of the same embryo, showing the spinal cord
and notocord again single as they are in regions more anterior than fig. 19.

THE AMERICAN JOURNAL OF ANATOMY, VOL 10, NO. 3.

386 Charles R. Stockard.

tic anesthetic effects, but they are strikingly common in the
aleohel solutions. On the other hand, similar abnormalities of
the nervous system are really infrequent in the weaker Mg solu-
tions.

The slight effects of Mg on the development of the central
nervous system are interesting when compared with the marked
effects of other anzesthetics on these tissues. In this regard it is
important to remember that in physiological experiments on nerve-
muscle preparations Mayer (’08) has found that Mg acts directly
as an inhibitor of muscular activity, exerting little if any effect on
the activity of the nervous parts. The action of Mg in these
experiments is not particularly on the nervous system but more
largely on the dynamic processes concerned in the outpushing of
the eyes. -

The occurrence of the several ophthalmic anomalies is common
to all of the anzesthetic solutions and similar conditions have not
been found in embryos treated with various salts and sugars
(06, ’07) which may inhibit general development and induce
many other abnormalities.

THe Errects oF CHLORETONE, ETHER AND CHLOROFORM ON
DEVELOPMENT

Chloretone, ether and chloroform when employed in weak
solutions influence developing eggs in a way somewhat similar
to that described for aleohol. The action of these substances is
not so pronounced and may be described more as an inhibition of
the general developmental processes. The embryos are usually
small and recover very slowly from the inhibiting effects after
being returned to sea-water. A few of the individuals in all
these solutions exhibit the cyclopean defect in its various degrees
just as it has been described in the specimens treated with alcohol
and Mg. .

The ophthalmic defects, eyclopia, monophthalmicum asymme-
tricum and entire absence of optic vesicles, are all conditions of
arrested or inhibited development and are prevalent among
embryos treated with solutions having anesthetic properties.

Effect of Aleohol and Anzsthetics. 387

The writer is led to conclude, therefore, that his former hypo-
thetical explanation of why cyclopia occurred in embryos treated
with Mg solutions was correct. The evidence strongly indicates
that the ophthalmic abnormalities produced in these experiments
are the result of an anesthetic action during the early develop-
mental stages.

THE PERIOD OF DEVELOPMENT AT WHICH CYCLOPIA MAY BE IN-
DUCED BY CHEMICAL AGENTS

The Mg experiments were repeated mainly to ascertain at
how late a period in development eggs could be subjected to the
solutions and subsequently develop into ecyclopean monsters.
The original experiments seemed to demonstrate the fact that
cyclopia was due to external influences acting on the development
of the optic vesicles, and not in any sense to a germinal variation.
Nevertheless, H. H. Wilder (08) in the face of these results,
advanced a germinal theory to account for the origin of cyclopia
and attempted to explain away the obstacle offered in the experi-
ments referred to by claiming that the eggs were subjected to the so-
lutions at so early a stage in development that germinal variations
might still have been induced. ‘This is impossible, asthe writer has
pointed out elsewhere (’09b), since germinal variations may only
be induced before embryonic development has begun. After
the two-or four-cell stage (the time at which the eggs were sub-
jected to Mg) is reached any thing done to the egg has its effect
on the developing embryo to cause this or that abnormal condition.
The only germinal variation possible at such a period would be in
the primordial germ cells of the developing individual, a variation
which would not manifest itself until the next generation of
individuals.

The following experiments prove beyond doubt that cyclopia
may be produced by the action of environmental influences.

When eggs in the eight-cell stage, or three and one-half hours
after fertilization, are subjected to the action of Mg solutions
many of the resulting embryos will show the cyclopean defect.

388 Charles R. Stockard.

If eggs be placed in Mg solutions five hours after fertilization,
when in the sixteen or thirty-two-cell stage, an almost equally
large number of cyclopean individuals will occur. The same is
true when they are subjected to the solutions after having devel-
oped in pure sea-water for seven and one ea hours and reached
the sixty-four or higher cell stages.

Eggs that have developed eleven hours in pure sea-water and
are in the early periblast stage with a somewhat flattened blast-
odermic cap, may be put into Mg solutions and caused to form
cyclopean monsters. The percentage of cyclopean embryos
arising from eggs treated at this late period is small, yet even after
developing for fifteen hours in pure sea-water some eggs may be
induced to form cyclopean monsters by treatment with MgCl, in
sea-water solutions.

Eggs that were older than this before being introduced into the
solutions failed to respond, all developing into ordinary two-
eyed individuals. This is due to the fact that a considerable
amount of time is necessary for the Mg to act upon the body sub-
stances of the early embryo and prevent normal eye development.
The optic vesicles begin to push out from the brain before the
thirtieth hour in development. ‘Thus, after the first six or seven
hours, the longer the eggs have been allowed to develop naturally
the smaller the proportion of cyclopean individuals that may be
artificially induced. After fifteen hours no embryo will be so
affected, since an insufficient amount of time exists for the Mg to
act on the eye anlagen.

The solutions are effective up to a stage in development pre-
ceding 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.

There can be no further doubt that cyclopean monsters are
caused by the action of a strange environment on the developing
fish embryos. With such evidence at hand it is also highly
probable that mammalian cyclops are due to the action of external
influences on the embryo and not to an abnormal germinal tend-
ency.

Effect of Alcohol and Anesthetics on Development. 389

OTHER CASES OF CYCLOPIA IN FISH

The Italian observer, Paolucei (74), has described a most
remarkable cyclopean ray. The monster was almost adult in
size, probably two years old, and measured 47cm. across the
pectorial fins and 20cm. in length not including the long whip-
liketail. Paolucci states that this cyelopean monster was captured
in the Adriatic Sea near the shore and had evidently been able
to cope with its surroundings and grow into a vigorous ray. So
far as the writer knows this case of a eyclopean monster in nature
being able to sustain itself and reach the adult stage, is unique.

Paolucei’s specimen proves the correctness of the writer’s
statement (’09) that the cyclopean eye is not necessarily associ-
ated with a single instead of a double brain, or with any other
serious defect in the brain region. This fact was clearly shown in
the brain structure of many of the cyclopean embryos studied,
as well as by their apparently normal behavior after hatching
from the egg.

The cyclopean Funduli have been kept living for more than one
month, which is as long as the experiment was tried. They would
doubtless have lived much longer, as they were hardy and able
to obtain an abundance of food from the vegetable particles in
the sea-water. Paolucci’s observation would indicate that the
Fundulus monsters might be reared to maturity and possibly
interbreed.

Gemmel (’06) has described four cases of cyclopia in newly
hatched trout collected from a fish-hatchery in England. The
conditions of the eyes and brains in these monsters are exactly
similar to those in the artificially produced Fundulus monsters.

The developing trout’s egg demands water of such high purity
that trouble is often experienced in the hatcheries, and monstrous
embryos commonly occur. These may result from weakened
developmental forces due to an insufficient oxygen supply or to
the accumulation of injurious chemicals about the eggs.

Gemmel (’06b) in describing cases of supernumerary eyes in the
trout embryos records that in one ease of an aborted twin head
the lens alone of all the eye structures was present. Free lenses

390 Charles R. Stockard.

were also described and figured in other individuals. Free lenses
oceur very commonly in heads showing various eye abnormalites;
a full consideration of these cases is recorded elsewhere.

SUMMARY

1. When the eggs of the fish, Fundulus heteroclitus, are subjected
during early stages of development to the action of weak solutions
of aleohol the resulting embryos show marked abnormalities in
the structure of their central nervous system and organs of special
sense.

The eyes in such individuals are either both small with poorly
differentiated retinee, cyclopean, asymmetrically monophthal-
mic or entirely absent. These ophthalmic defects sometimes
occur in as many as 98 per cent of the specimens. Such anomalies
are closely similar to those previously induced with Mg, and in
both cases are probably due to the anesthetic property of the
substances acting upon the eggs.

Alcohol tends to suppress the development and differentiation
of the auditory vesicles. A few specimens are entirely without
ears, others have one ear more or less perfectly developed while
the opposite ear is scarcely formed at all and still other individuals
have both ears extremely defective. In all cases examined the
better ear is invariably on the side with the better developed eye
if the eyes are also asymmetrically formed. The most persistent
portion of the internal ear, or that part which exists when all
other parts are wanting, is a cavity with an epithelial lning
resembling closely in structure an ampulla of the semicircular
canals. This fact may be interpreted to mean that the ampulla
is one of the most ancient or fundamental parts of the ear, and it
might further be considered indicative of the archaic function of
the ear as an organ of equilibrium since this is the chief function
of the ampulle. |

The brain is usually narrow and pointed in embryos that have
been treated with alcohol. It occasionally has a dorsal hernia
and shows regions of poor differentiation. The cell arrangement
jn the spinal cord are abnormal in many cases and spina-bifida

Effect of Alcohol and Anssthetics. 391

is not infrequent. These conditions of the central nervous sys-
tem might result from any cause that tends to retard development
and are not particularly characteristic of anesthetic solutions
as the eye anomalies are; yet the defects of the central neryous
system are commoner in these anesthetics than in any other solu-
tions with which the embryos have been treated.

2. Chloretone, chloroform and ether induce much the same
structural deformities in these embryos as does alcohol. They
act, however, as more general anesthetics, causing a retardation
in development. The characteristic eye and ear defects are not
nearly so common, though they do occur as a result of treatment
with these three substances.

3. The effects of Mg on the developing fish’s egg have been
previously considered. This substance is even more local in its
action than alcohol, the principal defects resulting from its use
being various anomalous conditions of the eyes, whereas the ner-
vous system generally may be in many cases structurally normal.
The embryos on hatching from the egg are able to swim in the
usual manner and live for more than one month in aquaria, which
is as long as any effort was made to keep them. The latter fact
would seem to indicate that the nervous system also functionates
normally.

Magnesium was used to test at how late a period in develop
ment the eggs might be introduced into the solutions with the sub-
sequent development of the cyclopean condition. It was found
that after normal development had proceeded for two, four, six,
eight, ten, eleven, twelve or even fifteen hours, if the eggs were
then placed in MgCl, solutions, many of the resulting embryos
showed the cyclopean defect. At fifteen hours the eggs have
reached the periblast stage and the blastodem is flattening down
upon the yolk. The germ ring arises shortly after this time and
begins its downward growth over the yolk mass.

Whenever eggs are allowed to develop beyond the fifteen hour
period before being introduced into the solutions of MgCl, they
invariably give rise to normal two-eyed individuals. The occur-
rence of cyclopia is less frequent when eggs are subjected at later
stages than when introduced into the MgCl, solutions during the
four or eight-cell stage. This is doubtless due to the fact that a

392 Charles R. Stockard.

considerable period of time is necessary for the Mg to act upon the
substances of the embryo and influence the origin of the optic
vesicles. When an insufficient time intervenes between the
period at which the eggs are subjected to the action of the solu-
tion and that at which the optic vesicles are given off from the
brain the Mg is unable to influence the tissues so as to induce the
eyclopean condition.

The production of cyclopia by the action of Mg at such late
stages in development proves beyond doubt that this deformity
is due to the action of external or environmental conditions on
the developing animal. Any explanation of cyclopia based on
germinal hypothyses such as that recently advanced by H. H.
Wilder must be reconstructed so as to conform to these facts-

Accepted by the Wistar Iastitute of Anatomy and Biology January 12, 1910. Printed June 21, 1910.

BIBLIOGRAPHY

GemmeEt, J. F. On cyclopia in osseous fishes. Proc. London Zoél. Society, i, pp.
1906 443-449.

1906b Notes on supernumerary eyes, and local deficiency and reduplication

of the notocord in trout embryos. Proc. London Zoél. Society, i,

pp. 449-452.
Harrison, R. G. Embryonic transplantation and the development of the ner-
1908 vous system. Anat. Record, ii, pp. 385-410.
Lewis, W. H. Experimental evidénce in support of the theory of outgrowth
1907 of the axis cylinder. Am. Jour. Anat. vi, pp. 461-472.
1909 The experimental production of eyclopia in the fish embryo (Fun-

dulus heteroclitus). Anat. Record, 11, pp. 175-181.
Mau, F. P. A study of the causes underlying the origin of human monsters.
1908 Jour. Morph. xix, pp. 1-361.
Mayer, A. G. Rhythmical pulsation in seyphomedusae. Carnegie Institute of

1908 Washington, Pub. No. 102, pp. 113-131.
Paouuccr, L. Sopra una Forma Mostruosa della Myliobatis Noctula. Atti della
1874 societa Italiana di Se. Naturali. xvii, pp. 60-63.
Srockarp, C. R. The development of Fundulus heteroclitus in solutions of lith-
1906 ium chlorid, with appendix on its developmentin fresh water. Jour.
Exp. Zodl., iii, pp. 99-120.
1907 The influence of external factors, chemical and physical, on the
development of Fundulus heteroclitus. Jour. Exp. Zoél., iv, pp.
165-201. 2
1909 The development of artificially produced cyclopean fish, ‘the mag--

neslumembryo.”’ Jour. Exp. Zodl., vi, pp. 285-338.
1909b The origin of certain types of monsters. Am. Jour. Obstetrics.
lix, no. 4.
Wiper, H. H. The morphology of cosmobia. Am, Jour. Anat., viii.pp. 355-
1908 440.

THE INDEPENDENT ORIGIN AND DEVELOPMENT
OF THE CRYSTALLINE LENS

CHARLES R. STOCKARD

From the Department of Anatomy, Cornell Medical School, New York City

WITH TWENTY-EIGHT TEXT FIGURES AND TWO PLATES

BR PHDioc +. 2s en pepe os oa) 4 Re ee 393
0 SL ST -RIDG ERS S| | err oo ie ee 396
PRETO CMOTYVOS............. 0... +6 See ee elie mine said Sel sie 397
PAS MMICUTTSPCLION........... ... .. 2. «sos pean eee. - aaa 2 400

a Is the origin of the lens from the ectoderm dependent upon a contact
Ma TOmiae OptIC VESICIE?. . ..; .../..- ace ee Treion eee ons. = 400

b Is the lens-plate or lens-bud capable of differentiating into a lens with-
Spmeaataciwadwan Optic CUP?.......2.-2ssee sche eesenes sched ese: 401

c Does the size of the optic cup regulate the size or shape of its associated
CELE). ¢2 2:25, 52 re ope so, co oe se eee eee 404

d Is the optic vesicle, normal or defective, always capable of stimulating
lens formation from the ectoderm at some stage of its development? ... 405

e May the optic vesicle cause lens-formation from ectoderm other than
aawmmnortmally forms alens?........ S:-cnteeeeees oe seeee so--s- 408

f Does a deeply buried eye have the power to regenerate or form a lens
MP EMNIEBICS ©, .... . . «a 2s sees Ones ate le cate ae 410
5. Discussion of previous observations and experiments..................... 412
MVeTMNCORENISIONS. ......... .:. ssa. Saue aeteeriee toes oc obs cece 419
7 oS Ue reniny 2S Pee so a ot oP) oes Pee 421

INTRODUCTION

The crystalline lens normally arises in the embryo from ectoderm
overlying the optic vesicle and continues its development in
close association with the optic cup. This fact suggests that
some correlation exists between the manner of development of
the optic cup and the optic lens. In ease such a correlation does
exist, to what extent is the optic vesicle and cup responsible for
the origin and subsequent development of the lens; and, on the
other hand, what influence, if any, does the presence of the lens

394. Charles R. Stockard.

exert over the development of the optic vesicle into the cup and
its subsequent development-into the eye?

If it is proven that the optic vesicle and cup possess the power
to derive a lens from the ectoderm the further question then
arises is the ectoderm under any condition able to give rise to a
lens without the optic vesicle stimulus? If so, is this independent
lens-bud capable of self-differentiation to such an extent as to
form a perfectly constructed lens?

Many questions of detail, as, for example, the relationship
between the size of the optic cup amd the size of the lens, the
ability of partial or defective optic cups to stimulate lens forma-
tion, whether the lens may be derived only from certain areas
of ectoderm or from any ectoderm, and other problems which we
will presently consider, also present themselves.

The study of these questions has come to be known as the
lens problem. Experimenters have attacked the questions from
several sides during the last ten years and the lens problem has
in many ways been beautifully analysed, yet today much addi-
tional evidence is necessary to entirely clear the situation.

In a previous paper the writer (’09) briefly described the inde-
pendent origin and self-differentiation of the optic lens in the
fish embryo. Since that time further experiments have yielded
much additional material and evidence bearing on this subject.
The results of these experiments are considered in the present
contribution and show in a most convincing manner that the
crystalline lens is capable of originating independently from the
ectoderm and of subsequent self-differentiation. Definite proof
will also show that although the lens may arise independently,
nevertheless the optic vesicle invariably has the power to stimu-
late the formation of a lens from any overlying ectoderm with
which it may come in contact.

The action of the optic vesicle on the ecioaers is a much
stronger force for the production of a lens than is the innate
tendency of the ectoderm to produceanindependentlens. Slghtly
injured ectoderm may be rendered unable to form a free lens
while the same weakened ectoderm will respond to a contact

Independent Development of the Lens. 395

stimulus of the optic vesicle by forming a lens. This point is
important, for herein the writer believes, after a study of his
own experiments and the results of other workers, lies the explana-
tion of many discrepancies in the operation experiments on the
lens. When the operation is so performed that the ectoderm
must be folded away in order to extirpate the optic vesicle and is
then returned to its place, free lenses have failed to occur, although
an optic vesicle may still have been able to derive a lens from
this replaced ectoderm. On the other hand, when the early open
medullary plate is operated on so as to remove the optic vesicle
areas, the ectoderm of the head wall is sometimes left uninjured
and from it may arise free lenses. The free lenses of King’s
experiments arose in embryos which were operated on dorsally
_ to burn out the optic vesicle areas of the partially open medullary
tube. The lateral head ectoderm was probably uninjured in
some of the specimens. In Spemann’s more recent experiments
the early open medullary plate was operated upon directly to
remove the optic vesicle areas; in such experiments free lenses
arose from the uninjured ectoderm. Experiments on other spe-
cies at such stages and in a similar manner will probably give like
results.

In the experiment of removing the optic cup and leaving a par-
tially differentiatied lens, this lens may have degenerated or
ceased to differentiate on account of the injury it suffered by the
operation, the absence of the optic cup not affecting it. It is
unquestionably true that in some amphibians and fishes the lens
is capable of perfect self-differentiation.

The optic cup does not exert complete control over either the
size or shape of the optic lens. Numerous points of detail are
also elucidated by the study of the optic organs in artificially
produced fish monsters which are either blind or present various
eye defects.

The experimental part of this investigation was conducted dur-
ing the summer of 1909 in the Marine Bioligical Laboratory at
Woods Hole, Mass., while occupying one of the rooms of the
Wistar Institute.

396 Charles R. Stockard.

2 METHOD AND MATERIAL

In all former experiments on the developing lens, except
those which the writer recorded (’09), mechanical methods have
been resorted to in destroying the early optic vesicle. This has
been accomplished by burning the region with hot needles or by
cutting away the tissue. It matters not how cleverly such
experiments may be performed they are often open to objection,
particularly when the experimenter has to remove or injure the
overlying ectoderm in order to reach and extirpate the optic
vesicle.

The present experiments have been conducted in an entirely
different manner. When developing fish embryos, Fundulus
heteroclitus, are treated with certain magnesium salts, alcohol,
chloretone or other anzsthetic agents, the development of the
optic vesicles is prevented entirely in some cases, while in other
specimens only one vesicle forms on either the right or left side,
and finally a large majority of the embryos present the cyclopean
defect with a-more or less double ventro-median eye. We have
here, therefore, an exceptioal opportunity to study the relation-
ship between the development of an optic vesicle and a lens. In
the first case, does a lens or do lenses ever occur in the eyeless
specimens? Does a lens ever appear on the eyeless side in the
single-eyed monsters? Finally do lenses ever arise in their usual
lateral positions when the embryo has a ventro-median cyclopean
eye? Allof these propositions are affirmatively answered without
an operation to injure in any way the ectoderm or tissues in the
primary lens-forming region. It may be thought that the action
of the chemical or anzesthetic is as severe as an operation but
this is probably not true, as the embryos after being treated for
the necessary time with magnesium, on being returned to sea-
water develop, hatch and swim actively about, living in aquaria
as long as I have tried to keep them, more than one month.

The experiments of the past summer have convinced the writer
that his former idea that the eye defects are due to the anzesthetic
properties of magnesium is correct; and there is no reason for
believing that certain tissues usually between the eyes are entirely

Independent Development of the Lens. 397

absent. These results are given in full in-another paper. The
solutions employed act as anesthetics preventing the usual out-
pushing of the optic vesicles from the brain to a greater or. less
degree, and give exactly the same condition so far as contact
influence of the optic vesicle on the lens is concerned, as though
the optic vesicle was cut away, without the disadvantages
accompanying the operation.

The experiments with anesthetics furnish arichness of material,
hundreds of specimens being obtained, which one would be unable
to duplicate from operations without spending days of tedious
labor. The crystalline lenses may be seen with the binocular
microscope as spherical refractive bodies in the living specimens.
The experimenter in this way is enabled to select various condi-
tion for study.

The embryos were best preserved for histological study in
picro-acetic though many fixitives gave good results. The lenses
stain equally well in eosin or picric acid used as a counter stain
after Mann’s hematein or Delafield’s hematoxylin.

3 THE LENS IN LIVING EMBRYOS

The living embryos at various stages when examined with a
binocular microscope show lenses isolated entirely from the optic
vesicle or optic cup. Figure 1 illustrates an embryo eighteen
days old that has no trace whatever of optic cups yet two per-
fectly developed transparent lenses occupy almost normal posi-
tions on the sides of the head. Sections of this specimen show the
two lenses with well differentiated fibers, fig. 5 and plate II,
fig. 8. Mesenchymatous tissue les between the lenses and the
brain. We must conclude that these lenses arose in lateral
positions and continued development and differentiation with no
direct influence whatever from either optic vesicle or the brain
tissue. The possibility of optic cups having arisen and degener-
ated is entirely out of the question, since, in the first place, this
has never been known to occur in any of the hundreds of Fundulus
embryos that the writer has studied. In the second place, plate I,
fig. 1, shows a lens arising from ectoderm on the eyeless side of

398 Charles R. Stockard.

a seventy-six hour embryo; it is scarcely conceivable that an
optic vesicle arose at about the thirty-fifth or fortieth hour,
came in contact with the ectoderm and entirely disappeared,
leaving no trace Of itself by the seventy-sixth hour. Further,
the lens in these cases must continue to develop and differentiate
independently, which is contrary to the position held by Spemann
(05), Lewis (’07) and LeCron (’07) for the frog and salamander.

Fig. 2 shows a fish nineteen days old with two well formed
lenses of normal size lying in contact with two small irregularly
shaped masses of heavy black pigment. In sections, figs. 23 and
24. the lenses are found to be perfectly formed and the two dark
spots are shown to be masses of choroid tissue or retinal pigment
without a definite retina or other associated eye parts. The lenses
may owe their existence to the choroid spots but the latter have
failed to influence the size or manner of development of the former.
If the origin of these lenses was due to the influence of the choroid
areas we have a striking example of how very small an amount of
optic tissue may call forth a lens. Many instances will be given
to show that extremely small amounts of optic tissue touching
the ectoderm will stimulate a lens to form, yet these cases will in
no way weaken the evidence that lenses do at other times form
entirely independently.

A fish is illustrated by fig. 3 with two small defective eyes
deeply buried in the head. The right eye possesses a lens but
the left faces ventrally into a mass of mesenchyme and is without
a lens. In front of the left eye is shown a lens in an extremely
anterior position but completely separated from the eye, and the
concave surface of the cup is not directed towards this lens. Fig.
4 proves the case by showing two slightly small eyeseach possess-
ing its own lens, while somewhat in front and between the two
eyes lies a perfectly isolated and independent lens. It is evident
that this supernumerary: lens is independently formed and not
due to a stimutus trom either of the eyes, since each has its
own lens.

Independent Development of the Lens. 399

3

CamerA Drawrinas or Living FunpuLus Empryos, MAGNIFIED 16 TIMES

Fie. 1 An embryo eighteen days old. First thirty-six hours after fertiliza-
tion were spent in 7 per cent alcohol in sea-water. No optic cups formed, yet two
perfect crystalline lenses are shown in the sides of the head. See fig. 5 for a sec-
tion of these lenses.

Fig. 2 Anembryo nineteen days old, first thirty-six hours in 9 per cent alcohol.
Two perfect lenses near poorly formed eyelike choroid spots. See figs. 23 and 24
for sections of these lenses and eye spots.

Fig. 3 Anembryo of same age and lot as fig. 2; afree lens is seen in an extremely
anterior position in front of an‘ll-formedeye. Lens. L.

Fig. 4 Another embryo of the same lot, showing a free lens between two some-
what defective eyes, each of which centainsitsownlens. Sections of thisembryo,
figs. 9 and 10, show also a second free lens which was hidden in the living specimen.

400 Charles R. Stockard.

4 THE EMBRYOS STUDIED IN SECTION

a “Is the Origin of the Lens from the Ectoderm Dependent upon a
Conlact Stimulus from the Optic Vesicle?

Spemann (’01) and Herbst (01) first introduced the view that
the lens originates from ectoderm only when a contact stimulus
from the optic vesicle is present, although Spemann (’07) has
since modified his opinion for one species of frog at least. Lewis
(04 and ’07) has found this to be true in his experiments on frog
tadpoles, and LeCron (07) on salamanders. King (’05) claims,
however, that such is not the case in her experiments and holds
the view that the lens arises independently of the contact stimulus
by the optic cup. Lewis (’07) has brought objections to the
method employed by King and so criticises her results, but the
writer believes her method has a real advantage in that she burnt
out the optic vesicle areas of the still open medullary tube from the
dorsal side and thus may not always have injured the lateral
ectoderm of the future lens-forming region.

The writer’s experiments on the fish embryos clearly demon-
strate that the origin of the lens from the ectoderm may be entirely
independent of the contact stimulus of the optic vesicle. He
continues to use the expression ‘‘contact stimulus of the optic
vesicle”? since this is what has been deemed necessary for the
origin of the lens, although he believes that a lens may arise with-
out any stimulus whatever from the optic vesicle either by con-
tact or from a distance.

In specimens lacking optic vesicles entirely it is difficult to
imagine that tissues are present in the brain which possess the
power to formsubstances characteristically formed by optic vesicles
and that these substances diffuse until they reach the ectoderm and
stimulate it toforma lens. In the ease of isolated supernumerary
lenses the optic cups possess lenses but still other lenses arise at
a distance.

Again referring to fig. 1 of plate I, a section through the eye
region of a seventy-six hour embryo, the ectoderm on the eyeless
side is forming alens which is somewhat slower in development

Independent Dee of the Lens. 401

than the lens in the eye on the other age yet this bud shows dis-
tinct lens character.

A perfect lens is seen in fig. 2, plate I, to be entirely separated
from the brain and no optic cup exists. Fig. 13 shows a lens in
a small choroid cup and a second free lens lying near. Fig 7
illustrates a similar case. Figs 8 and 12 show extremely anterior
lensesin eyeless individuals and again fig. 5and fig. 3,plate II, show
two beautiful lateral lenses in another eyelessspecimen. Fig.6shows
a section with three well differentiated lenses all free from contact
with an optic vesicle; a more posterior section of this embryo,
fig. 27 shows a choroid cup deeply buriedin brain tissue and with-
out alens. This cup does not come in contact with either of the
three lenses shown in the more anterior sections.

Finally, a most remarkable case of supernumerary lenses is
illustrated by figs. 9 and 10 and the outline fig. 11 shows the
position of these lenses in the entire head (see also plate II,
fig.4). Two defective eyes each possessing a lens areshown in sec-
tion, fig. 10, and a third lens lies between the eyes. In a more anter-
ior region, fig. 9 and plate IJ, fig. 4, is found another section of this
third lens, C, and a fourth additional protruding lens lies below it.

These cases might be enumerated and illustrated until they ran
into the scores, but sufficient evidence has been given to prove
that the crystalline lens in these embryos does not depend upon
a contact stimulus of the optic vesicle for its origin from the ecto-
derm but originates independently.

6 Is the Lens-Plate or Lens-Bud Capable of Differentiating into a
Lens without Contact with the Optic Cup?

The above question is convincingly answered in the affirmative
by the evidence given in the foregoing discussion. In the older
embryos it is clearly shown that supernumerary lenses are as
highly differentiated and as perfectly formed in all respects as
are the lenses in the eyes. LEyeless individuals, as figs. 1 and 5
and plate II, fig. 3, indicate, may possess perfectly formed trans-
parent ——— which appear in the living Boconeen 8 as clear refrac-
tive bodies.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 10, No. 3.

402 Charles R. Stockard.

SECTIONS OF WELL DIFFERENTIATED FREE LENSES

Fic. 5 Asection of two free lateral lenses in the nineteen day embryo shown by
fig. 1.; no trace of optic cups exists. This embryo has two ears of unequal size.

Fia. 6 Section of the head of nineteen day embryo, first thirty-six hours in 9
per cent alcohol. ‘Three free lenses, A. B. and C are shown, a defective optic cup
completely separated from the lenses is deeply buried in the head tissues of amore
posterior region, see fig. 27.

Fig. 7 Section of the anterior tip of the head of a nineteen day embryo from 9
per cent alcohol. The upper right lens protrudes from a defective eye shown in
more posterior sections, while the lower lens, F., is free, being in no way associated
with an eye.

Fra. 8 A somewhat sagittal section of a similarly treated embryo of same age,
showing another free lens, p, a pigment spot.

Fic. 9 An anterior and fig. 10 a more posterior section through the eye region
of a nineteen day embryo. treated with alcohol. The diagram, fig. 11, shows the
plane of both sections. Fig. 4 shows the same embryo from life; the eyes in the
sections are reversed by the microscope. ‘Two optic cups are present each with a
lens, A.and D, while two other perfectly differentiated lenses, B and C, are not
connected with an optic part.

Fig. 12 Asection through the anterior tip of a pointed-headed eyeless embryo
nineteen days old. The lens is well differentiated; the ears in this specimen are
scarcely formed.

Fic. 13 A section of a nineteen day embryo showing a small defective choroid
cup with a lens, and a second accessory lens is near by.

403

Independent Development of the Lens.

404 Charles R. Stockard.

The possibility of the action of some substance given off by a
distant optic cup is entirely aside, since other experinienters have
claimed that when the optic vesicle or cup is in any way separated
from the lens the latter organ begins to degenerate and usually
disappears.

Figs. 6, 7, 9, 10, 12, 13 and 21 and plate I, fig. 2, plate II,
figs. 3 and 4, all go to show tkat in the fish embryo the lens-plate
or lens-bud is capable of self-differentiation, finally forming a per-
fectly transparent refractive body even though completely isolated
from any other eye-like structure.

c Does the Size of the Optic Cup Regulate the Size or Shape of tts
Associated Lens?

Lewis (’07) has stated in his more recent paper on the lens that,
“The lens is neither self originating nor self differentiating, but
is dependent for its origin, its size, its differentiation and its
growth on the influence of the eye.’? The writer had also inde-
pendently been led to think from his first experimental study of
eyclopia (’07), which was based on a limited supply of material,
that the size of the lens varied directly with the size of the optic
cup. He is now able to show that while normlly the lens and
optic cup are properly adjusted as to size this is not by any means
constantly true of ill-formed eyes. Here the size and also the
shape of the lens is often greatly out of accord with that of the
optic cup. In normal eyes the optic cup has a definite size and
so does the lens. The sizes accord, yet this may be incidental or
entirely without correlation, as is suggested by the fact that optic
cups of unusual shape and sizeare not able to regulate the develop-
ment of the lens so as to adjust it to their strange proportions.

Many of the illustrations of eyclopean eyes given in the writer’s
recent paper (09) show misfits between the cups and lenses.
Remarkable cases are also shown by figs. 9 and 10 and plate II,
figs. 4 and 5, in which the lenses are clearly too large for the asso-
ciated cups. Fig. 15 shows the two components of an incomplete
cyclopean eye with one normally proportioned lens between them.
This lens is scarcely large enough to function with the unusually

Independent Development of the Lens. 405

wide double cup. In fig. 20 is seen a somewhat similar double
cup with an elongated and slightly constricted lens; fig. 18 also
shows one half of a double cyclopean eye with a double lens,
the other half eye is in a more posterior section. In figs. 14 and 17,
on the other hand, we find illustrated double lenses in one of
two closely approximated eyes. Fig. 14 also shows a tiny addi-
tional lens still further within the same cup which possesses the
large double lens. Fig. 16 shows the extreme anterior tip of a
cyclopean eye with two minute lenses protruding from it. This
section is only fifty micromillimeters from the anterior end of the
head. Numerous other examples of misfitting lenses might easily
be given.

These facts force us to conclude that the size of the optic cup
does not fully regulate either the size or shape of the associated
lens. It is, therefore, evident that the usual harmonious adjust-
ment between the optic cup and the lens may not be so entirely
due to a dominating influence of the optic cup on the lens as one
might be led to believe from previous contributions to the sub-
ject. That some influence or interaction exists, the writer does
not deny, and will show in the following parts of this paper the
remarkable ability possessed by the optic vesicle to obtain a lens
from any part of the ectoderm with which it may come in contact.

d Is the Optic Vesicle, Normal or Defective, always Capable of
Stimulating Lens-Formation from the Ectoderm at Some Stage
of its Development?

Of all the embryos which the writer has examined not one failed
to have a lens in a normal optic cup when the cup came in con-
tact with the ectoderm. If, however, the cup fails to reach the
outer body wall, although it may possess well differentiated reti-
nal layers and other parts, it is invariably without a lens, fig. 26.
The convex side of the choroid coat or pigment layer of the retina
does not cause a lens to arise even though it be closely applied to
the ectoderm, as is shown by fig. 26 and many other illustrations
in which the choroid touches the body wall.

Defective optic cups when deeply buried and separatea from

406 Charles R. Stockard.

Itt-ADJUSTMENT OF Optic Cups AND LENSES IN THIRTEEN Day FisH EMBryos.

Fic. 14 A section of an embryo treated with 5 per cent alcohol. Both optic
cups are defective, the left one contains a spherical lens, while the right cup has
a large lens, in shape a constricted oval, and a tiny spherical lens placed fur-
ther within the same cup. H, ear.

Fic. 15 A poorly formed eye of the incomplete cylopean type. Each com-
ponent faces in a ventro-median direction and a spherical lens lies between them
An ear, E. is shown on the side of the better component, the other ear is absent.

Fic. 16 A-section 50 microns from the anterior tip of anembryo. The anterior
border of a cyclopean eye is shown with two protruding lenses of very minute size.
ec, ectoderm.

Fira. 17 Section through the anterior tips of two small closely neighboring
eyes, whose median planes are in more posterior sections. The smaller eye has a
protruding double lens. This embryo possesses only one ear located on the side
with the better eye.

Fic. 18 Part of an incomplete cyclopean eye (other half in more posterior
sections) containing a double lens.

Fie. 19 Two adjacent eyes facing ventro-medianly with two lenses.

Fie. 20 An ovoid lens in an incomplete cyclopean eye.

——

407

Independent Development of the Lens.

C= a

—

Ss

=>

(

(

4

408 Charles R. Stockard.

the ectoderm also lack a lens, as is illustrated on the left of figs.
27 and 28. On the other hand, it is remarkable how small and
ill-formed an optic cup-like structure has the power of stimulating
a lens to arise from the ectoderm. Figs. 2, 23 and 24 and plate
II, fig. 5, show small choroid cups with no retinal differentiation
whatever, yet closely associated with perfectly formed lenses. In
fig. 21 is seen an extremely defective cup with a small lens; a
larger independent lens is shown on the eyeless side. Fig. 22
illustrates an extremely insignificant eye-like body buried within
the brain, yet close by is a small crystalline lens; these are the
only eye parts found in thisembryo. In fig. 19, a section through
the eye of an incomplete cyclops, each component of the eye has
a lens, while in fig. 20 the eye components are closer together and
in fig. 15 further apart, yet each of these possesses only a single
lens, although it is elongate in fig. 20. It is difficult to say why
such eyes occasionally possess two lenses. After an examination
of a large number of such eyes no general rule is found. It may
be due in some way to the manner in which the optic cup periph-
ery meets the ectoderm, whether as a circle, an oval or at times
a much constricted oval so that two areas of ectoderm are sepa-
rately stimulated to form lenses.

This consideration forces the conclusion that an optic cup at
some stage in its development, whether normal or defective,
invariably possesses the power to stimulate lens-formation from
the ectoderm with which it comes in contact.

e May the Optic Vesicle Cause Lens-Formation from Ectoderm
Other than that which Normally Forms a Lens?

This question is answered by the cyclopean monsters. It is
scarcely conceivable that the ectoderm which would normally lie
over the lateral eyes has the power to migrate, or follow the optic
vesicle so exactly as always to lie just over the vesicle wherever
it may chance to develop. Many embryos have eyes in unusually
anterior positions and derive their lenses from anterior ectoderm,
while others possess ventral eyes with lenses derived from ventral
ectoderm. It occurs in a few cases, as the writer previously

Independent Development of the Lens. 409

EXTREMELY DerectivE Optic Cup-Like Boptes ASSOCIATED WITH PERFECT

LENSES

Fie. 21 An anterior section, as is indicated by its size, of a nineteen-day em-
bryo. A free lens is shown on the left while on the right a very small defective
eye contains a small lens.

Fie. 22 Asmall lensnear a vesicle-like structure, E, in the brain which might
represent an abortive optic body.

Figs. 23 and 24 Sections through the embryo shown in fig. 2. In the diagram,
fig. 25, is shown approximately the planes of the sections. The large well-formed
lenses are associated with defective optie cups which lack any sign of differentia-
tion.

410 Charles R. Stockard.

recorded (’09), that free lenses arise in their usual lateral positions
while the cyclopean eye possesses its lens of anterior origin.

In the fish embryo the optic vesicle may cause a lens to form
from ectoderm far removed from the usual lens-forming area,
and rarely in such cases it happens that free lenses may also
arise from the usual lens-forming region.

f Does a Deeply Buried Eye have the Power to Regenerate or Form
A Lens from its own Tissues?

It has been shown ky many experimenters Colucci (’91), Wolff
(95 and ’01), Miller (96) and Fischel (’02), that the salamander’s
eye regenerates a new lens from the posterior surface of the iris
when the old lens is removed, or as Fischel found, if the old lens
be merely pushed back out of its usual placeinthe eye. When the
iris was injured in two places during the extirpation of the lens,
two lenses arose within the single eye, one growing from each
injured area of the iris. It has also been found that a fish’s
eye would regenerate a lens under certain conditions: when the
fish is young and when a sufficiently long time is allowed for the
lens to regenerate.

Lewis (’04) finds that the deeply buried eyes in Rana palustris
which fail to come in contact with the ectoderm are unable to
form lenses from their own tissues. While on the other hand he
states that in a second species, Rana sylvatica, the optic cup
readily gives rise to a lens from its own tissues if prevented from
stimulating the formation of a lens from the ectoderm of the body
wall.

The fish embryos which are now being considered, act in a
similar manner to Rana palustris and are unable to form lenses
from the tissues of their optic cups. Whenever the optic cup
is deeply buried and fails to reach the ectoderm it also fails to
possess a lens, as is illustrated on the left side of figs. 26, 27 and
28. In this connection it may be mentioned that Morgan was
unable to obtain the regeneration of a lens in adult specimens
of Fundulus, although as mentioned above, lenses do regenerate
in the eyes of another species of fish.

Independent Development of the Lens. 41]

99 60)

O00
04

.)

A oa
=. C) os

‘ 3
Sar

28

DEEPLY BuRIED EYES witHout LENSES

Fig. 26. <A section of a thirteen day embryo which was treated with alcohol.
One eye faces outward and contains a lens, while the other faces in toward the
median plane of the head and is without a lens, probably never having come in
contact with ectoderm.

Fig. 27 A section of a nineteen day embryo more posterior in the same series
than section, fig. 6. The deeply buried defective eye has no lens.

Fig. 28 Section of thirteen day embryo after treatment with alcohol. Sec-
tion passes in front of the median plane of the right eye which is well differentiated
and contains alens. The other eye is small and scarcely differentiated, buried in
the tissue below the brain and contains no lens. A normal ear exists on the side
with the large eye and a small defective ear is on the other side in more posterior
sections.

412 Charles R. Stockard.
5 DISCUSSION OF PREVIOUS OBSERVATIONS AND EXPERIMENTS

Before drawing final conclusions from the cases discussed above
it may be well briefly to review the modern work and experi-
ments which have been directed towards a solution of the so-
called lens problem.

Rabl (’98) in his study of the structure and development of the
lens, found in one case, at least, that a lens-like body was present,
although far removed from the optic vesicle. This observation
has been criticised by Lewis, who suggested that the ectoderm
with the newly forming lens had shifted away from the small
optic vesicle. Again, Lewis states that Rabl’s case, and also a
case to be considered below that was shown by Mencl, prove
neither one side-nor the other, since the experimental evidence
is all directly for the idea that a lens will not arise from the skin
without the stimulus of the optic vesicle. At the present time,
however, the experimental evidence points in an opposite direc-
tion, and the cases of Rabl and Mencl can scarcely be disposed
of in so brief a manner. On the contrary Rabl’s example must
be considered the initial illustration of the origin of an early
lens without a stimulation from the optic vesicle.

Following this single case a strong tide turned towards the
idea of the dependent origin and development of the optic lens.
Herbst’s paper (’01) on ‘‘Die formative Reize in der thierischen
Ontogenese”’ and Spemann’s pioneer experiments (’01) on the de-
velopment of the lens seemed most convincing evidence in favor of
a correlation in development between the optic vesicle and the
lens, a correlation in which the latter played a dependent role.
Herbst claimed the optic lens to be a ‘‘Thigmomorphose”’ origi-
nating only by a contact between the optic vesicle and the epi-
dermis. His reason for such a position being that in the case
of cyclopean monsters the median optic vesicle always derives
a lens from the overlying ectoderm, while no lenses arise in the
usual lateral positions. “If the lens is an independent organ why
does it not arise in the lateral eye region of cyclopean monsters?”
In former experiments the writer (’09) produced just such a case
as Herbst thought necessary to show the independent origin of

Independent Development of the Lens. 413

lenses, that is, a cyclopean monster with lenses in the usual
lateral positions.

After a study of a great number of such monsters I am now able
to reaffirm that free lenses do occasionally arise in the lateral
eye regions. Herbst’s clever argument based on pathological
embryos is, therefore, rendered invalid.

Spemann (’01) was the first to clearly attack the problem experi-
mentally. He injured or destroyed the optic vesicles of Triton
embryos by means of hot needles and electric cauterizers. The
method was not altogether satisfactory since such an operation
often injures much of the surrounding tissue, yet Spemann’s
results were of the highest value, and stimulated an active interest
on the part of many experimenters. His conclusions furnished
strong support for Herbst’s idea of the dependent origin of the
lens. He found that whenever the optic vesicle was so injured
that it failed to come in contact with the head ectoderm, the
ectoderm failed to form a lens. The lens was, therefore, depend-
ent for its origin on a contact stimulus of the optic vesicle upon
the ectoderm. Later Spemann (’05) also concluded that the lens
was not self differentiating but that a durable contact stimulus
of the optic cup was necessary for it to form lens fibers. Thus
the ‘‘Herbst-Spemann theory of dependent lens formation,”
as Mencl has termed it, was developed. We shall see below,
since itseems best to consider these papers in a more or less chrono-
logical order, that Spemann’s own later work has helped materi-
ally to overthrow this theory.

Barfurth (’02) also operated witha hot needle to destroy the lens
anlage and the optic cup. The embryos were examined after
five or six days. One specimen showed on one side a poorly
regenerated optic cup that did not come in contact with the ecto-
derm and yet a lens still connected with the ectoderm was present
on this side. Barfurth was inclined to accept the Herbst-Spe-
mann idea of lens formation and so attempted to harmonize his
case with the theory as follows. Some sections of the embryo,
9 to 12, showed the optic vesicle lying very near the ectoderm
but not in contact with it. Barfurth supposes that at an earlier
period in development it may have been in direct contact with

414 Charles R. Stockard.

ectoderm and at such a time stimulated the lens to arise. This
is merely a conjecture and the more recent experiments of King
(05) and Spemann (’07) on the frog, and the writer’s on the fish,
would warrant equally well an interpretation of independent
origin for this lens.

Returning to observations on monsters we may consider the
case reported by Mencl (03) which has called forth so many
explanations and criticisms. This report described the existence
of an independent lens on each side of the head in an anadidymus
embryo of Salmo salar which he had possessed since 1899, his
interest in it being aroused by the contributions on the dependent
origin of the lens. One of the lenses in this monster was closely
applied to the brain wall, and in fact lay in a depression in the side
of the brain; the other lens, however, was completely separated
from the brain by mesenchyme. Spemann and Lewis have tried
to explain this case by assuming the existence in the brain wall of
some optic vesicle tissue or substance which when brought in
contact with the head ectoderm possessed the power to cause
the formation of a lens. There was absolutetly no evidence of
optic tissue in this brain wall and further, the explanation could
apply only to one of the lenses, though it scarcely explains the
origin of either. The second lens, entirely free and with mesen-
chymatous tissue separating it from the brain wall, was as perfect,
though not so large, as the other which lay against the brain.
This lens probably arose and developed freely, just as did so many
lenses in the fish embryos the writer has studied.

Mencl (’0S) has more recently obtained other anadidymus
Salmo embryos and confirms his former observations, finding that
in such monsters free lenses are present in 25 per cent of the cases.

Gemmill (’06) has also recorded the frequent occurrence of free
lenses in the heads of monster trout embryos. é

Schaper (’04) in considering a case of a typical lens develop-
ment comes to the theoretical conclusion that the lens is by
nature a primitive sense body similar to the sinnesknospe of
Amblystoma, and has secondarily taken on its present function.
It must, therefore, arise independently of the optic vesicle,
with which it is only recently associated. Such speculation

———————————————

Independent Development of the Lens. 415

is not entirely out of accord with the present facts of lens
formation. ;

Lewis thinks that the free rudimentary lenses in Schaper’s
experiments were undoubtedly caused by the shifting of the ecto-
derm and lens away from the optic vesicle. The writer is unable
to see any reason why such a shifting is supposed to have taken
place. On the contrary, the facts seem to lend themselves more
readily to the interpretation of a free origin of the lens.

The experiments of Lewis (’04) seemed to show convincingly
that the lens was dependent for its origin and development upon
a contact stimulus of the optic vesicle on the ectoderm. Lewis
devised a method far more refined than any previously used in
similar experiments. He operated on tadpoles under a binocular
microscope with needles and small scissors and was able to cut
the ectoderm and fold it forward so as to expose the brain and
early optic vesicle, which could now be cut away. The ectoderm
was then folded back in place. From these experiments the follow-
ing are some of the conclusions which were drawn:

Neither a lens nor a trace of a lens will originate from the ecto-
derm which normally gives rise to one, if the contact of the optic
vesicle with the skin is prevented.

There is no predetermined area of the ectoderm which must
be stimulated in order that a lens may arise. Various parts of
the skin when stimulated by optic cup contact may and do give
rise to a lens.

In normal development the lens is dependent for its origin
and differentiation on the contact influence or stimulation of the
optic vesicle on the ectoderm.

The conclusions are clearly stated and the experimental method
employed is most skillful, but not entirely free from objection.
The conclusion regarding the entire dependence of the lens on
an optic vesicle stimulus will not, the writer believes, be supported
by future experiments, and indeed at the present time such an
idea has a vast amount of evidence against it, unless the partic-
ular species experimented with shows a specific action in lens
formation. The later work of Spemann (’07) which contradicts his
former conclusion shows that in some species of frogs the lens

416 Charles R. Stockard.

may arise and differentiate independently of the optic vesicle
stimulus. In these experiments it must be noted that Spemann
operated with glass needles to remove the optic vesicle regions
from the open medullary plate. Such an operation does not
injure the lens-forming region of the ectoderm as Lewis’ experi-
ment does and it is on this account, the writer believes, that the
free lenses arise.

The tendency of the ectoderm to form a lens independently
is so delicately adjusted that a very slight injury or disturbance
may suppress. it, yet the same ectoderm may still have the power
to form a lens in response to the stronger stimulus of the optic
vesicle. So in experiments where the ectoderm has been cut or
injured it loses the power to form free lenses even though the optic
. vesicle can stimulate a lens to arise from it. When the ectoderm
is uninjured, as in some of King’s specimens, Spemann’s (07),
and the writer’s, then free lenses do occur.

We may imagine the lens to represent an ectodermal organ
formerly of independent importance. However, it has now be-
come so closely associated with the nervous portions of the eye
that it arises whenever such a part meets the ectoderm, yet the
lens retains to a feeble degree its impulse to arise independently
of other eye parts. When it has once arisen it is perfectly capable
of differentiation. Future experiments of removing the optic
vesicle without injuring the ectoderm will probably demonstrate
further this tendency of the lens to arise independently, just as
Mencl’s observations and the writer’s experiments show for the
fish, and many of the experiments mentioned show for amphibians.

King (05) states that she had begun her series of experiments in
1900. She destroyed the optic vesicles from the forebrain region
in the closing meduilary tube with hot needles. Many of the
embryos died as a result of theoperation. Theconclusionsreached
by King are entirely opposed to those of Lewis and Spemann’s
earlier results regarding the dependent origin of the lens. King
found that in some of the embryos a lens-like body arose from the
ectoderm on the side with an injured or unregenerated optic cup
which did not come in contact with either the ectoderm or the
lens-like body. Some of the specimens show a very suggestive

Independent Development of the Lens. A417

lens-like structure which is entirely free from any contact with
an optic vesicle. The inaccuracy of the operation may account
for these lenses, on the ground that in exceptional cases the outer
ectoderm of the lens region was too far distant at the early stage
of development to have been injured. This is probable when it is
noted that the operation did not enter through the lateral ecto-
derm, but through the partly open medullary tube from above.
The present evidence also goes to strengthen King’s position on the
subject.

Spemann (’05), Lewis (07) and LeCron (’07) have all claimed
that the lens after its origin, was not self differentiating but
depended upon a durable contact with the optic cup for its
future development. All of the writer’s experiments and the
observations of Mencl are clearly contradictory to such a view,
and Spemann (’07) has more recently found the lens to be self-
differentiating in Rana esculenta.

In a former paper the writer (’07) described the development. of
the lens in the blind Myxinoid, Bdellostoma stouti. At that time
he presumed that this animal furnished support for the experi-
mental evidence that the lens was not self-differentiating. In
Bdellostoma embryos the optic vesicle is well formed at first and
reaches out from the brain to the ectoderm; at this time the ecto-
derm forms a localized thickening suggesting a lens-plate. The
optic vesicle then ceases to maintain its progressive development
and loses connection with the ectoderm, finally giving rise to
the poorly formed optic cup of the adult. The early lens thicken-
ing of the ectoderm degenerates and is entirely absent from older
embryos. ‘The writer suggested that this degeneration was due
to the loss of contact with the optic vesicle, since such reasoning
seemed correct in the light of the experiments up tothattime. He
has entirely changed his view, however, regarding the matter of
the lens in Bdellostoma and now believes that it represents a
rudimentary organ which has been lost in the adult and appears
only for ashort time during embryonic life. As Eigenmann (’01)
records of Amblyopsis, the lenses of other blind fishes appear at
certain stages in the developing embryo and then degenerate. <A
case not so far advanced in the degeneration of the eye and lens is

THE AMERICAN JOURNAL OF ANATOMY, VOL. 10, No. 2.

418 Charles R. Stockard.

that of the Florida burrowing lizzard, Rhineura. In the adults of
Rhineura, Eigenmann finds lenses sometimes present and sometimes
absent and when present they are very variable. Somany examples
are known of the traces of rudimentary organs in the embryo
which, are entirely lacking in the adult that the above cases of
the lens are not at all strange and at present it seems that this
is the most satisfactory explanation of their behavior.

The more recent experiments of Spemann (’07) so frequently
referred to, may be briefly described. . Embryos of Rana escu-
lenta were operated upon with glass needles so as to remove the
optic vesicle areas from the open medullary fold (his former
experiments were made with hot needles, which doubtless injured
more of the surrounding tissues).

After allowing these embryos to develop for several days and
then studying them in section it was found that in one case on the
side of the head lacking an optic vesicle a true lens-bud was still
in connection with the ectoderm. In an older embryo a free lens
vesicle was found, and finally, in a still older specimen a lens was
buried in connective tissue on the eyeless side of the head yet
it possessed fully formed lens-fibers. Thus, Spemann, five years
after his first paper on the dependent origin of the lens, now con-
cludes that the lens in Rana esculenta is self-originating and
self-differentiating. He also accepts Mencl’s case of the inde-
pendent lens in Salmo salar.

We have thus seen in a rather cursory survey how the lens
problem has arisen and how experiments have built up first one
side of the question and then the other, and many may also feel
that the final word is yet to be added. Nevertheless, it is true
that at this stage of the investigation it has been clearly demon-
strated in several groups of animals that the ectoderm possesses
the power independently to originate a lens which subsequently
develops into the transparent refractive organ usually found
within the mature eye. The manner of origin and differentiation
of the lens may easily differ in different animal species, and the
statement would not be warranted that all of these facts apply
to animals in general.

The lens is self-originating and self-differentiating yet it is more

Independent Development of the Lens. 419

emphatically proven that the optic vesicle or optic cup has the
power without exception to cause a lens to form from the ecto-
derm with which it comes in contact, even though this ectoderm
may be far distant from the usual lens-forming area. There is
probably no strictly limited region of ectoderm from which the
optic vesicle must stimulate lens-formation. The self-originating
lenses, however, have invariably occurred in the head region,
though not always in their usual lateral positions. It would,
therefore, seem that the ectoderm of the head is more predis-
posed to the formation of lenses than that of the other body
regions.

6 SUMMARY AND CONCLUSIONS

1 When the developing eggs of Fundulus heteroclitus are
subjected to the action of Mg salts, alcohol, or other anesthetic
agents, the normal outgrowth of the optic vesicles is generally
inhibited. Embryos are obtained either entirely without optic
cups, with small deeply buried eyes, with only a single eye on one
side of the head or, finally, with a median more or less double
eyclopean eye. These specimens furnish exceptional material
for a study of the relationship between the development of the
optic vesicle and the optic lens. The embryos with the nervous
eye parts entirely lacking are similar to specimens with the optic
vesicles mechanically cut out of the brain. At the same time,
the injury to the ectoderm which usually results from the opera-
tion is avoided, and this is a most important advantage. When
Spemann (’07) operated on Rana esculenta embryos with open
medullary plates he was able to remove the optic vesicle areas
from the future inner side of the tube without injuring the ecto-
derm of the lens-forming region and in such cases independent
lenses arose on the eyeless side. King (’05) obtained free lenses in
a somewhat similar experiment. Lewis’ embryos might possibly
respond in a like manner if operated upon in this fashion. The
optic vesicle may stimulate a lens from slightly injured ectoderm,
but the free origin of lenses is a more delicate process and one more
easily interfered with.

490 Charles R. Stockard.

2 The crystalline lens may originate from ectoderm without
any direct stimulus whatever from an optic vesicle or cup. These
self-originating lenses arise from regions of ectoderm that are
not in contact with either optic vesicle, the brain wall, or any
nervous or sensory organ of the individual (see plate I).

3 The lens-plate or lens-bud is capable of perfect self-differen-
tiation. No contact at any time with an optic vesicle or cup,
is necessary. These lenses finally become typical transparent
refractive bodies exactly similar in histological structure to that
in the normal eye (see plate II).

4 The size and shape of the lens is not entirely controlled by
the associated optic cup. Lenses may be abnormally small for
the size of the cup, or entirely too large, so that they protrude;
or, finally, peculiarly shaped oval or centrally constricted lenses
may occur in more or less ordinarily shaped optic cups. The lens
is by no means always adjusted to the structure of the optic cup
as has been claimed by some observers.

5 An optic vesicle or cup is invariably capable at some stage
of its development of stimulating the formation of a lens from
the ectoderm with which it comes in contact. It is remarkable
how extremely small an amount of optic tissue is capable of stimu-
lating lens formation from the ectoderm (plate II, fig. 5).

6 The optic vesicle may stimulate a lens to form from regions
of the ectoderm other than that which usually forms alens. This
is shown by the fact that a median cyclopean eye always stimu-
lates a lens to form from the overlying ectoderm. It is scarcely
possible that the lateral normal lens-forming ectoderm could
follow the cyclopean optic cup to the many strange situations
it finally reaches.

7 The ectoderm of the head region is more disposed to the
formation of lenses than that of other parts of the body, since the
free lenses invariably occur in this region.

8 A deeply buried optic vesicle or cup may fail to come in
contact with the ectoderm; in such cases it lacks a lens. The
tissues of the embryonic cup itself are unable to form or regenerate
alens. This is not true in all embryos, as Lewis has shown for
one species of frog.

— alee

"a. .

Independent Development of the Lens. 421

9 The optic lens may be looked upon as a once independent
organ (possibly sensory or perhaps an organ for focusing light on
the brain wall, before the vertebrate eye had arisen) which
has become so closely associated with the nervous elements of
the eye that it has to some extent lost its tendency to arise inde-
pendently, although still capable of doing so under certain con-
ditions. The lens now arises much more readily in response tu a
stimulus from the optic vesicle, a correlated adjustment which
insures the almost perfect normal accord between the optic cup
and the lens. In experiments on the origin of the lens one must
guard against disturbing the ectoderm from which an independent
lens might arise. Although an optic vesicle might have the power
to stimulate a lens from injured ectoderm, the innate tendency of
the ectoderm to form a lens is a more delicate impulse and may be
suppressed by a slight injury or disturbance of the ectoderm
during the operation.

7 BIBLIOGRAPHY

BarrFurtH,D. Versuche iiber Regenerationdes Auges und der Linse beim Hiihner-
1902 embryo. Verh. d. Anat. Gesellsch. Halle.

Cotucctr, V.S., Rigenerazione parziale del!’occhio nei Tritoni. Mem. Accad. Se.
1891 Bologna, Series 5, Vol. i.

EIGENMANN, C. H. The History of the Lens in Amblyopsis. Proc. Indiana
1901 Acad. Se., 1901.

FiscHet, A. Weitere Mitteilungen iiber die Regeneration der Linse. Arch. f.
1902 Entw’Mech., Bd. xv.

GemMILL, J. F. Notes on Supernumerary Eyes, and Local Deficiency and Re-

1906 duplication of the Notocord in Trout Embryos. Proc. Zodl. Soc.
London, Vol. i.
Hersst, C. Formative Reize in der thierischen Ontogenese. Ein Beitrag zum
1901 Verstandniss der thierischen Embryonalentwicklung. Leipzig,
H. Georgi.
Kine, H.D. Experimental Studies on the Eye of the Frog. Arch. f. Entw’ Mech.,
1905 XIX.
LeCron, W. L. Experiments on the Origin and Differentiation of the Lens in
1907 Amblystoma. Am. Jour. Anat., vi.
Lewis, W. H. Experimental Studies on the Development of the Eye in Am-
1904 phibia. I. On the Origin of the Lens. Rana palustris. Am.

Jour. Anat., ili.
1907 Experimental Studies, etc. iii. On the Origin and Differentiation of
the Lens. Am Jour. Anai., vi.
Accepted by the Wistar Institute of Anatomy and Biology January 12, 1910. Printed June 21, 1910.

422 Charles R. Stockard.

Menct, E. Ein Fall von beiderseitiger Augenlinsenausbildung wahrend der
1903 Abwesenheit von Augenblasen. Arch. f. Entw’ Mech., xvi.
1908 Neue Tatsachen zur Selbstdifferenzierung der Augenlinse. Arch. f.
Entw’ Mech., xxv.
Mixirr, E. Regeneration der Augenlinse nach Extirpation be Tritonen. Archiv
1896 f. Mikrosk, Anat., xlvii.
Ras, K. Ueber den Bau und die Entwickelung der Linse, I. Zeitsch. f. Wiss-
1898 Zool., Bd. 63. ;
Scrarer. A. Uber einige Fille atypischer Linsenentwickelung unter abnormen
1904 Bedingungen. Ein Beitrag zur Phylogenie und Entwickelung der
Linse. Anat. Anz., xxiv.
SpemMANN, H. Ueber Correlationen in der Entwickelung des Auges. Verhandl.
1901 der Anat. Gesellsch., 1901.
1905 Ueber Linsenbildung nach experimenteller Entferung der primdren
Linsenbildungzellen. Zoél. Anz., xxiii.
1907 Neue Tatsachen zum Linsenproblem. Zodél. Anz., xxxi.
Srockarp, C. R. The Artificial Production of a Single Median Cyclopean Eye
19074 in the Fish Embyro by Means of Sea-Water Solutions of Magnesium
Chlorid. Arch. f Entw. Mech., xxiii.
19076 The Embryonic History of the Lens in Bdellostoma stouti in Relation
to Recent Experiments. Am. Jour. Anat., vl.
1908 The Question of Cyclopia. Science, n. s. xxvill.
1909 The Development of Artificially Produced Cyclopean Fish, ‘‘The
Magnesium Embryo.” Jour. Expr. Zodl., vi.
Wourr, G. Regeneration der Urodelenlinse. Arch. f. Entw.’—Mech., I und xii.
1895 and 1901.

Accepted by the Wistar Institute of Anatomy and Blology January 12, 1910. Printed June 21, 1910.

ee

Independent Development of the Lens. 423

EXPLANATION OF PuaTEs I anp II

Fig. 1 A photograph of a section of a seventy-six hour Fundulus embryo; the
brain is almost bilateral and perfect, with a well formed left optic cup, no indica-
tion of the right cup exists, yet the ectoderm on that side has formed a well
pronounced lens-bud, L.

Fig. 2 A section through the eye region of a thirty day embryo, there is no
optic cup in the specimen. A perfect lens is in the usual lateral position, L, a
band of muscle, M, lies between it and the brain.

Fie. 3 A section through the head of a Fundulus embryo eighteen days old.
The specimen is shown in text-figure 1, no eyes are present, yet two perfectly differ-
entiated crystalline lenses are seen in the sides of the head. A close examination
of the photograph will reveal mesodermal cells between the lens and the brain.

Fig. 4. A section through one defective eye which possesses a lens of dispro-
portionate size, while on the other side of the head two free lenses are seen, the
lower small one protruding from the head. A second defective eye with a lens
is found in a more posterior region of the head. Text-figure 4 represents this
embryo as it appeared in life.

Fic. 5 A section through one of the very defective optic vesicles shown in

‘fig. 2. The photograph illustrates the extreme lack of proportion which may

exist between the size of the optic vesicle and its associated lens. It indicates
also how small an amount of optic tissue may stimulate lens formation from the
ectoderm.

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INDEPENDENT DEVELOPMENT OF THE LENS PLATE I
C. R. Stockarp

THE AMERICAN JOURNAL OF ANATOMY, VOL, 10, No. 3.

PLATE II

INDEPENDENT DEVELOPMENT OF THE LENS
C. R. StocKakp.

‘
ee ee ~

THE AMERICAN JOURNAL OF ANATOMY, VOL. 10, NO. 3.

Studies of Tissue Growth.

Ill. The Rates of Regenerative Growth in Different Salt Solutions.
1V. The Influence of Regenerating Tissue on the Animal Body.

By

“

Charles R. Stockard,

Cornell University Medical School, New York City.

With 4 figures in text.

Eingegangen am 18. Oktober 1909.

lll. The Rates of Regenerative Growth in Different Salt Solutions.

The changes in the various physiological actions of the body
induced by changing the inorganic salt constituents of the blood
suggests that such inorganic salts may also be of importance in deter-
mining the rate and manner of growth. Logs ('05) has shown that
regenerative growth does not take place at all in the absence of the
K ion. Beese (04) found that rapidly growing malignant tumors
contained an excess of K while benign tumors showed excesses of
Ca. An excess of Ca, however, is usually present in old or degen-
erating tissues and so may accumulate during inactivity instead of
being the cause which produces the inactive state. Lor (04) also
tried the influence of dilute sea water on regenerating hydroids and
found the hypotonic solution to cause a more rapid rate of regener-
ation than occured in normal sea water. He further tried some of
the inorganic salts with indefinite results.

In the first of this series of studies I ('08) recorded the action
of the four important metalic ions of sea water, Na, K, Ca, and Mg,
on regeneration in the medusa. The experiments were not extensive
enough to draw final conclusions yet they accorded with what might
have been expected from the related work of Lors and Breese men-

16 Charles R. Stockard

tioned above. The Na ion retarded regeneration, and in some solutions
of CaCl, regeneration did not begin for several days and always
proceeded slowly, while in the weaker KCl solutions regeneration
occured at a more rapid rate than in the control specimens. The Mg
solutions gave indefinite results.

It must be remembered that in such experiments the entire animal
is kept immersed in the solution. The effects of the salt may, there-
fore, be systemic exhilarating or depressing the entire animal body
and through such conditions secondarily affecting the regeneration
rate. Should salts be injected into the circulation of higher animals
the same complexity presents itself, yet this fact in no way lessens
the importance of such experiments since all chemical actions and
processes in the body secondarily effect other parts than those in
which they occur. The experimenter, however, must carefully guard
against using a dose of the salt which would be sufficient to per-
minantly weaken or injure the body since this would necessarily
lessen the rate of regeneration as well as other normal processes.

The present series of experiments were arranged in order to
carry regenerating animals for long periods of time in strange salt
solutions and so. determine whether there was any definite effect on
the regeneration rates, and if such an effect was sufficiently marked
to be of advantage in experimentation. The spotted salamander,
Diemyctylus viridescens, was used for the experiments since it readily
regenerates new legs.and tail after the old ones are amputated. This
gives two somewhat different structures for observation the legs being
complex while the tail is almost uniformly metermeric. ‘The experi-
ments were conducted for the Huntington Fund for Cancer Research
in the Pathological Laboratories of Cornell Medical School.

Five groups each consisting of fifteen salamanders were selected
and weighed. The groups A, B and D each weighed 39 grams and
groups C and E weighed 38 grams thus the groups were practically
of the same average size. The individual salamanders averaged
about 2.5 grams and were closely alike in size and general body
condition. The operations consisted of cutting the left front leg off at
the elbow joint and the distal one-third of the tail (Fig. 1A). The
tails were measured from the posterior end of the cloacal apature to
the tip, the usual length was from 45 to 48 mm. making the removed
third measure from 15 to 18 mm.

The first few days following the operation the arm stump is
generally held close to the body and not used in swimming or walk-

-
od

Studies of Tissue Growth. III. ity;

ing upon the bottom of the vessel. After this time the stump is
used occasionally at first but later it is brought into continual use.

The vessels containing the salamanders were kept upon the same
table and subjected to similar light and temperature conditions. The
animals were fed on alternate days with finely chopped beef each
individual receiving approximately the same amount.

Fig. 1.

A. A salamander illustrating the operation
in the first part of the experiment. Left
fore-arm amputated at the elbow and one-
third of the tail cut away.
B. The operation in the second part of the
experiment. Right fore-arm amputated at
the elbow and the regenerated tail part cut
away. Regenerated tissue stippled.

The experiments are to be considered in two parts; the first
from Oct. 9th to Dec. 22nd, 73 days, while arranged as described -
below and the second part a consideration of the groups is a reversed
order in the solutions, e. g., those in KCl were put into CaCl, ete.

First Part.

Three days after the operation the animals were placed in the
following solutions. Group A, the control, in ordinary tap water.
Group B into MgCl, 2/;.; m, or 16 ce of a molecular solution of MgCl,
to 984 ec of tap water. Group C in CaCl, 2/,25 ™, group D in KCl
2/49, m and group E into a solution of 8 ce molecular MgCl, + 8 ce
molecular CaCl, added to 984 ce of tap water. The solutions were
changed daily.

Archiv f. Entwicklungsmechanik. XXIX. 2

18

Mable I.
Average records of regeneration from salamanders kept in salt solutions.

(o

Charles R. Stockard

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So) os! Se coe CoCo ars
Rl SS Fe sh COCO cS
a] & = | 2
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eat 3 | Qi tig | co &
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- a tS el
a| es & te R Serene.
|S YF] ip ee | OO
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an od |
: eB |
ech esi |) SE 22 Se) |) —
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aa to 5 (>. a Liggt
A) Siac pe See eevee
| M1929 | ©
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= | Ss 5 Sk CO 19 co CO
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Las | Sitesi aS
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mH ip se A i = :
S S) e0"F || seacs) OG) |e bao
aoa) st = = =
aa 3
Fe
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me Se | ete es
DO) Bee a aS eee >
z |= ane eee
= 7 Aaagq|iAaar
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|

The effects of these salts were
tested on other salamanders. It
was found that a ‘/,) 7 solution
of either KCL, CaCl, or MgCl,
would kill the animals in less than
two days. The strengths used
were, therefore, about one sixth
of the fatal dose but no appreci-
able injury to the veneral body
health could be detected in such
solutions.

Thirty-five days after the oper-
ation the newly regenerated tissue
and body lengths were measured.
The averages for each group are
shown in table I. The average
body lengths are almost equal, or
little different in consideration of
the entire length. The control is
regenerating the leg and tail faster
than any other group at this time.
The figures 8D, 3D ete. in the
leg-bud columns indicate the
number of leg-buds which show
differentiation or the formation of
toes. Those groups in KCl and
MgCl, + CaCl, are almost up with
the control in the lengths of new
regenerated buds while the group
in MgCl, is slower and the spec-
imens in CaCl, are slowest of all.
It is recalled that the mixture of
MgCl, and CaCl, consists of only
one half as much of each salt as
is used in the unmixed solutions
and while both of these salts have
retarded regeneration in the strong
unmixed solutions the mixture has
not lowered the rate.

The next line of the table shows that after fifty-two days. the

x

Studies of Tissue Growth. III. 19

relative body lengths have remained about the same. The new leg-
buds in KCl are now much longer than the control, about 25 °/).
Those in the MgCl, + CaCl, have also overtaken the control, while
on the other hand, the specimens in MgCl, are behind the control
and those in the CaCl, are still further behind. The tail-buds in
KCl and MgCl, + CaCl, have also grown faster than the control
since the thirty-fifth day. ‘Those salamanders in MgCl, have kept
their original slow rate while the tail-buds in CaCl, like the arm-
buds in this solution are the shortest of all, the tail-buds particularly
have fallen still further behind since the thirty-fifth day.

Seventy-three days after the operation the arm-buds are in much
the same relative condition, while the tail buds of the control have
grown more rapidly than those of the other groups. The CaCl,
group being slowest.

Since the experiment started one individual in each group has
been lost through accident, the groups now consist of fourteen indi-
viduals each. At this time, Dec. 22nd, the groups were again weighed.
The lines below allow a ready comparison with their original weights.

Groups A B C D E
Control MgCl, CaCl, KCl MgCl + CaCl,
Oct. 9 39 er. Sol ee en ooer, 39) ar: 38 er.
Dee. 22 41.4 45.2 44.1 46.1 47.3

The original weights were for the fifteen individuals in each
group while the lower line for the fourteen thus shows a steady
gain in weight during the experiment due no doubt to the regular
feeding. It will be noted by referring to table I that the increase
in weight and body length of the groups does not correspond, e. g.,
group A has increased very much less in weight than group E while
on the other hand group E has increased less in body length than
group A. The increase in weight in these salamanders is due to an
enlargement of the body organs rather than to a storing up of fat.
The safer criterion in determining actual growth is the increase in
body length rather than increase in weight since the later fluctuates
so readily, and as MorGan ('06) states a normal rate of regenerative
growth is kept up even though the body be in a thin emaciatea
condition.

The general result of the experiment thus far seems to show
that the salamanders are capable of regeneration while living in the

salt solutions. The specimens in the KCl regenerate at a rate equal
Ox

20 - Charles R. Stockard

to and often ahead of that shown by the control, while those in
CaCl, and MgCl, are decidedly inhibited in their rates of regenerat-
ing both legs and tails. ‘These facts accord with what might be ex-
pected from Brrse’s chemical analysis of tumors referred to above,
but we must look further in the experiment for final results.

After the seventy-third day the animals were put into pure water
so as to remove all excess of the several salts to which they had
been subjected. After being in the water for twenty-four days the
groups were again measured and the results are shown in the 97
day line of table I. The measurements stand in much the same re-
lation to one another as that existing while they were in the solu-
tions, this might have been expected since the time is scarcely suf-
ficient for the effects to have been entirely overcome. After the salts
had been thoroughly removed the animals were again operated upon
and treated as recorded in the

Second Part.

The five groups, now consisting of fourteen salamanders each,
were operated upon so as to remove the right front leg at the elbow
joint and the entire new tail-bud (Fig. 1B). The arm operation gives
a new cut for regeneration while all of the tail operations give rise
to a second regeneration from the same level as that from which
the previous new tail-buds arose.

Jan. 22nd, four days after the operation the animals were put
into the salt solutions so that the groups which were in the retard-
ing solutions (MgCl, and CaCl.) during the first part of the exper-
iment were now put into the solutions which had seemed to accelerate
regeneration (KCl) and visa versa.

Group A (Control) now Control.

Group B (MgCly 2/,95 m) now KCl 2/;95 2m.

Group © (CaCl, 2/42; m) now KCl 2/195 m.

Group D (KCl 2/;95 m) now CaCly 2/195 2.

Group E (MgCl, 4/19, m +-- CaCl, 1/,9; m) now MgCle */125 22.

A summary of the records of the animals in these solutions. is
shown in table IL.

After thirty-nine days the arm-buds are all short and no differ-
entiation has taken place. It will be noted that these buds are in
most cases only about one half the length of the thirty-five day buds
in table I. The tail-buds have regenerated at a rate nearer that

2
=
a

Studies of Tissue Growth. III. 21

shown in the first part of the
experiment. The groups that were

Table IT.

Kept in salt solutions in reversed order.

Average records of regeneration from the same salamanders as in table I.

aggq| =non
: B* 5 | Pane formally slow in MgCl, and CaCl,
2 | =, | 2228 are still behind the control al-
= 2 Ai i i, " though now in KCl. After sixty-
Blin uailiteat ~ico’. one days the right arm-buds are
2 pas (2.2. about equal to the length the left
_ 822 755 buds had attained at fifty-two days.
te Et cc In the first part of the experiment
= a= 2 FS = theD group in KC] was consider-
A oe aaaa- ably ahead of all the others, it
2 2 F | i if : ns has now lost this advantage in the
Eonar rio ac CaCl, solution. Groups B and C
2 | 4 2 | ae Poe which were originally regenerat-
ced ge SSS s ing behind the control in MgCl,
i Balog and CaCl, ee, ce
@\|s&+,| *=S"= to improve significantly in the
= = /aaaa solutions of KCl. Group E which
g 2 a1 b4 = — maintained a rate higher than the
o | 8 = SEES control when in a mixtnre of
2 eyes fee =a a is ie eed
22° &3523 - ahead of the control in MgCl,
1 ee alone although after this time it
|32 : san falls behind the control, yet re-
8 ; Ee ani generates faster than any other
5 Bs S = = S group in the salt solutions.
a | S82 | NQ9NH When in these solutions for
2 TS == seventy-eight days the control is
S 2% Be} 2 Sa” well ahead in length of the arm-
eee buds. Group B in KC] after having
222 ,2%5 formerly been in MgCl shows the
2 eed aaa poorest record and has only 8
g| 3 soaoo 7) leg-buds that have differentiate
2 2: as ne toes, while the other four groups
2 “| =+3S have well formed toes on almost
-s 232/022. all ofthe new legs. Group C which
ae “~~ “previously showed the weakest re-
2.8 Bios generation in CaCl, is now, while
Bigs ™ in the KCl solution, to be favor-

ably compared with any other

99 Charles R. Stockard

eroup in the salt solutions. Eleven of the fourteen leg-buds have well
differentiated feet and toes.

These results would seem to indicate that the influence a salt
exerts depends somewhat upon the former salts to which the animal
has been subjected, at any rate, in this case the KCl acts more favor-
ably when used after CaCl, than when following MgCl.

The tail-buds after seventy-eight days, like the legs, are slowest
in group B but groups C, D and E are ahead of the control.

The last line of the table which shows the condition after one
hundred days is not significant since growth in all the groups is very
slow and almost complete at this time, the processes of differentia-
tion being primary.

The animals were again weighed on Apr. 28, about seven months
after the experiment was started. The weights at the periods indi-
cated are given below.

Groups A B C D E
Conteh tat Bit G1 Key > eae MgCl,
Oct. 9 igh aly 39 er. 38 gY.! 30 sem 38 er.
Dee. 22 41.4 45.2 44.1 46.1 47.3
Apr. 28 46.5 44.5 Hie iS Ea.) 50.8

The increase in weight of the animals shows that they were in
a healthy condition and not suffering from the treatment. From
Dec. 22 to Apr. 28 group B lost slightly in weight and this is of
interest when it is remembered how very poorly the individuals of
this group regenerated in KCl after having formerly been treated
with MgCl. The evidence indicates that treatment with MgCl, pro-
duces a condition in the animals which renders them unfavorable to
treatment with KCl solutions.

A final point of interest to be noted in the reversed second part
of the experiment is the condition of the new legs in group ©. The
left first leg in five of the fourteen specimens had not differentiated
in the CaCl, solution to which they were subjected in the early part
of the experiment and actually after seven months two have legs
with conical terminations with one toe in one case, while three others
have only slight indications of differentiation. The right first legs
in these same specimens had been growing only during the second
part of the experiment (100 days) yet with a single exception they
were perfectly differentiated in the KCl solution. This fact shows

Studies of Tissue Growth. III. 93

in a decided way the different influences exerted by the CaCl, and
KCl solutions on two legs of the same individuals. The bottom line
of table I gives the measurements and differentiation of the left legs
after 199 days when the experiment ended, these may be compared
with the right legs in table II.

Summary.

Considering both parts of the experiment one may conclude that
the effects of the salt solutions are not strongly pronounced, yet the
following statements seem to be supported.

1) The processes of regenerative growth in Dremyctylus are
favorably affected by weak doses of KCl while CaCl, inhibits the rate
of growth and differentiation of the regenerating part.

2) Solutions of MgCl, inhibit both the rate of growth and dif-
ferentiation of regenerating parts, yet not so strongly as CaCh.
Mixtures of half doses of CaCl, and MgCl, do not influence either
growth rate or differentiation.

3) If salamanders having had their regenerating powers retarded
by MgCl, be then placed in KCl instead of this salt stimulating growth
it further depresses the regeneration of both legs and tails and little
if any differentiation takes place.

4) When specimens that had regenerated slowly in CaCl, were
placed in solutions of KCl their rates of regeneration and powers of
differentiation were improved. The effect exerted by a salt solution
is, therefore, dependent to some extent upon the salts to which the
animal has been previously subjected, even though some time may
have elapsed since the former treatment was applied.

5) Salamanders that have regenerated at a fair rate in solutions
of KCl are less depressed by treatment with CaCl, than others which
have not been previously treated with KCl.

6) All actions of salt solutions on the body of a regenerating
animal are probably very complex and the above statements are more
to be taken as suggestions than final conclusions; they may serve
best to indicate the need of extensive investigation along such a line
which may possibly open a way to the control of growth processes.

Zusammenfassung zu Teil III.

Wenn man beide Seiten des Versuches in Betracht zieht, so kinnte man
zu dem Schlusse kommen, da8 die Wirkungen der Salzlisungen nicht stark aus-
gepriigt sind; immerhin scheinen die im folgenden ausgesprochenen Siitze ge-
niigend gestiitzt zu werden:

24 Charles R. Stockard

1) Bei Diemyctylus werden die regenerativen Wachstumsprozesse durch
schwache Dosen von KCl in giinstigem Sinne beeinfluBt, wihrend CaCl, den
Wachstums- und Differenzierungsbetrag in den regenerierenden Teilen ver-
mindert.

2) Liésungen von MgCly vermindern sowohl den Wachstums- wie den Diffe-
renzierungsbetrag in den regenerierenden Teilen, aber nicht so stark wie CaCl.
Mischungen der halben Dosen von CaCly und MgClo beeinflussen weder den
Wachstumsbetrag noch die Differenzierung.

3) Werden Salamander, deren Regenerationsfihigkeiten durch MgClo ge-
hemmt sind,, nachtriiglich in KCl versetzt, so iibt dieses Salz anstatt seiner
sonstigen wachstumférdernden Wirkung eine noch weiter deprimierende auf die
Regeneration sowohl der Beine als der Schwiinze aus, und es findet wenig oder
gar keine Differenzierung statt.

4) Wenn Exemplare, welche in CaCly langsam regenerierten, in Lésungen
von KCl gebracht wurden, so wurden ihre Regenerationsgeschwindigkeit und
ihre Differenzierungsfihigkeit verbessert. Die von einer Salzlésung ausgetibte
Wirkung ist demnach in gewisser Ausdehnung abhiingig von der Art der Salze,
denen die Tiere vorher unterworfen waren, selbst wenn einige Zeit seit der
ersten Behandlung verstrichen sein sollte.

5) Salamander, die mit groBer Geschwindigkeit in Lésungen von KCl re-
generiert haben, erleiden durch Behandlung mit CaCly eine geringere Depression
als andre, welche vorher nicht mit KCl behandelt worden sind.

6) Alle Einwirkungen von Salzlésungen auf den Korper eines regenerie-
renden Tieres sind wahrscheinlich sehr kompliziert und die oben ausgesprochenen
Siitze miissen mehr als Vermutungen wie als endgiiltige Schliisse aufgefafBt
werden; sie kinnen aber gut dazu dienen, auf die Notwendigkeit ausgedehnter
Forschungen in einer Richtung hinzuweisen, die méglicherweise einen Weg zur
Beherrschung der Wachstumsvorgiinge erdffnet. Uiberetst Gebhardt.

IV. The Influence of Regenerating Tissue on the Animal Body.

When the adult animal body begins to regenerate new tissue in
order to replace some lost part or when abnormal secondary growths
arise the condition of growth equilibrium is disturbed and such a
disturbance is followed by changes which effect the usual physiolog-
ical condition of the body. The question arises whether the changes
following or accompanying normal regenerative growth are in any
way similar to those affects resulting from malignant or abnormal
growths. If one believes that cancerous formations are growths in-
duced by some derangement in the normal growth states and are
not of infectious origin then normal secondary growths, in some stages
at least, should effect the body in a manner somewhat similar to that
resulting from an active tumor growth. The emaciated or cachectic

Studies of Tissue Growth. IV. 25

condition of the body resulting from cancerous growths are not al-
ways attributable to toxins or products produced in the cancer and
taken into the circulation, but at times seem due to the excessive
approperation of nutriment by the rapidly growing tumor itself. The
malignant tumor continues to grow and so finally kills the body,
while on the other hand, the regenerating part although rapidly
growing at first gradually decreases in growth rate and begins to
differentiate and function thus diverting the energy previously used
in the growth processes until finally growth ceases when the body
has reéstablished its former condition.

I (09) showed in the second of these studies that the medusa-
disk of Casstopea xamachana decreased rapidly in size while regen-
erating new oral-arms and that the rate of decrease was faster in
those specimens regenerating the greater number of parts. In these
experiments a sourse of error might have existed since those spec-
imens with six or eight oral-arms removed have been deprived of
more reserve food held in the mouth arms than had the individuals
which lost fewer arms. I determined to control this possibility by
operating on medusae so as to remove the same number of oral-arms
from all and to increase the amount of new regenerating tissue in
some individuals by removing also a part of the disk. The specimens
were kept under identical conditions and were not fed during the
time of the experiment. Thus any difference in their responses is
due only to the additional amount of regeneration imposed upon those
individuals with the cut disks.

Emmet ('06) has contributed an observation which is most inter-
esting in connection with these experiments. He found that larval
lobsters when regenerating new legs molted after longer intervals
than normal individuals and increased in size at a rate sometimes
24°/, slower than the non-regenerating specimens. Most important
was his observation that when the removal of legs was not followed
by regeneration such specimens grew in size faster than the regen-
erating individuals and in most instances actually faster than the con-
trol. These observations clearly show that the process of regener-
ation itself and not the injury inflicted is responsible for the retard-
ation of growth in the regenerating lobsters.

The experiments here recorded were performed upon the seypho-
medusa, Casstopea xamachana, which is so abundant at the Tortugas
Islands, Florida. Healthy individuals of medium size were selected
and operated upon as described below.

26 Charles R. Stockard

In the first experiment two groups of twenty individuals of the
same average size were used. Group A had five of the eight oral-
arms cut from each specimen (Fig. 1). Group B also had five oral-
arms cut in a similar manner from each of the twenty individuals
and in addition each medusa had a peripheral strip cut from its
hody-disk which included one third of the circumference and extended
in radially beyond the oral zig-zag muscular layer shown in Fig. 2.
The specimens were then allowed to regenerate for thirty-four days
their disk diameters being carefully measured at intervals so as to
determine the difference in body decrease of the two groups.

Fig. 2.

Fig. 1. Casstopea samachana with five of its oral-arms amputated at their bases.
Fig. 2. Five oral-arms and one-third of the disk periphery removed.
Fig. 3. A medusa with all of its oral-arms and the central stomach mass cut away.

Table I shows the records for group A, the first column giving
the original disk diameters, the second column the diameters after
twelve days, the third column the lengths of the individual new arm-
buds regenerated during the twelve days. Column four gives the
diameters after twenty days and column five the lengths of the new
arm-buds at this time. Columns six and seven show the same after
twenty-eight days and columns eight and nine after thirty-four days
when the experiment stopped. A line of averages at the foot of the
table shows the general result.

Table II gives the same data for group B and a ready compar-
ison of the tables is facilitated by table III of averages.

The individuals of both groups averaged 81.5 mm in diameter
at the beginning of the experiment and after twelve days the spec-
imens of group A were 67.5 mm in diameter while those in group B
which were regenerating the disk tissue in addition to the five oral-
arms were only 64.3 mm in average diameter. In other words they
averaged 3.2 mm smaller than the ones growing only the five arms.

27

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Studies of Tissue Growth.

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Studies of Tissue Growth. IV. 29

After this time, however, group B did not decrease as rapidly since
the disk injury had almost completely regenerated; thus after twenty
days the A group was only 1.7 mm larger than B, after twenty-eight
days only 1.2 mm larger and after thirty-four days there was still
only 1.4 mm difference in average size.

Table iT.
Comparison of averages when medusae are injured to different extents
A summary of tables [ and IL.

Disk diameters in mm at stated intervals Length of arm-buds in mm

one After | After | After

Original

After | After After After After
Dia. 12 days | 20days | 28 days | 34 days | 12 days | 20 days | 28 days | 34 days
53.5 | 49.7 4.7 5. 6.3 6.9

815 | 643 | 876

|
A | 815 | 67.5 | 59.3
B

oy)
52.3 | 48.3 4.1 5.4 6.25 6.9

The experiment clearly shows that while the B group was regen-
erating the cut disk part in addition to the five oral-arms the indi-
viduals of B were decreasing in body size as a result of this ad-
ditional regeneration more rapidly than the specimens in A which
were regenerating only the five oral-arms. ‘The regenerating tissue
through an excessive capacity for the absorption of nutriment draws
upon the old body tissues and causes them to. decrease in size ina
manner very much as it may be supposed the rapidly growing tumor
imposes upon the substances of the surrounding body. It is certainly
clear that in both cases the growing tissue causes the old body to
become weak and emaciated while the growth itself continues in a
vigorous manner.

The above experiment is of further value in regard to the in-
fluence of the degree of injury on the rate of regenerative growth.
I have previously shown ('09) that the rate of arm regeneration in
this medusa is independant of the degree of injury as is also the
ease in the brittle-star, Ophiocoma riser; while Ophiocoma echinata
regenerates each arm the slower the greater the number of removed
arms. These results are contrary to the idea advanced by ZELENY
(05) that the greater amount of injury would be followed by a faster
regeneration rate.

The two groups of individuals A and B are each regenerating
five oral-arms but the group B is the more extensively injured since
a portion of the disk was also cut away. If the additional injury
or regeneration imposed upon the B group exercises any influence

30 Charles R. Stockard

over the rate of regeneration it should be seen by comparing the
rates of growth of the arm-buds in B with those of group A. The
right side of table III gives a ready comparison of the two groups.
Those specimens with the disk uncut or the less injured ones regen-
erated slightly more rapidly during the first twelve days but after
this time the rates were practically equal. These facts show in a
most convincing manner that more extensive injury to the medusa
fails to give an increase in the subsequent regeneration rates.

A second experiment differed somewhat in manner of operation
from the above yet the results are in perfect accord. Twenty-eight
healthy medusae were arranged in two groups of fourteen individuals
each and operated upon as follows. The specimens of group I had
all of their oral-arms and the central stomach mass entirely removed,
leaving only the medusa disk (Fig. 3). Such a preparation lives and
pulsates in a normal manner and regenerates new tissue to cover
over the central stomach space, then begins to bud new oral-arms
from this tissue until finally the medusa regains its normal organs
and parts. The central space is first covered by a thin veil of tissue
which tears repeatedly and reforms until it begins to thicken and
then the new arm-buds first appear. The regenerative growth is,
therefore, very vigorous from such specimens during the early part
of the experiment and later becomes much less. Group II was oper-
ated upon in the same manner as the specimens of group B in the
above experiment, five oral-arms and a part of the medusa disk were
cut away, Fig. 2.

Labie ny
Decrease in size of medusae regenerating different amounts
of tissue.
Disk diameters in mm at_stated intervals Length of arm-buds in mm
Group | _ a a | |
Original After After | After | After After After
Dia. | = Hayey _ deve | 28 days 14 days 22 days 28 cage
| |
88.6 55.3 51 |
63. 59 44 | 52 6

Table IV facilitates a ready comparison of the averages from
these specimens. The original diameters of the groups averaged
88.6 mm and 88 mm, after fourteen days group I was only 62 mm
in diameter while group IL was 69 mm, or 7 mm larger. After twenty-
two days they were 55.3 mm and 63 mm and after twenty-eight days

Studies of Tissue Growth. IV. 31

51 mm and 59mm. It will be noted however that group I ceased
to decrease rapidly after the first fourteen days when its rapid regen-
eration also ceased and from this time on it decreased almost as
slowly as group II, since in the last six days of the experiment it
lost only 4.3 mm while group II lost 4 mm.

The rate of growth for the arm-buds in group II is practically
the same as from the specimens in the previous experiment.

Groups I and IL again show that when the animal regenerates
a certain amount of tissue in a given time such an individual suffers
a loss in body size which is greater than the loss from other spec-
imens regenerating a less amount of tissue. Of course the animals
must be subjected to the same food conditions, in these experiments
they were all unfed. Regenerating tissue, therefore, consumes the old
body substance and has an effect that would finally so weaken the
body as to cause death should the regeneration continue for a suf-
ficient time. A method which could eliminate the factors that cause
growth to cease when an organ attains a certain size would allow
the organ to grow at the expense of the other body parts until death
would follow in a manner closely similar to that by which a malignant
tumor growth finally kills the body containing it. The absence of
certain growth inhibiting substances in the body may be responsible
for indefinite cancer growths, and experiments that in any way lead
to a determination of the controling factors in normal primary or
secondary growths are of importance in this regard.

Summary.

The medusa, Casstopea xamachana, when unfed decreases in body
size. This decrease is greater in regenerating individuals, and the
larger the amount of tissue an individual is regenerating the more
rapidly does it decrease in size. The new regenerating tissue grows
at a vigorous rate on account of its excessive capacity for the ap-
properation of nutriment from the old body tissues, and it is this
fact which causes the body to decrease in size and become weak
and emaciated. A close similarity to such an action is seen in the
case of certain malignant growths.

Zusammenfassung zu Teil IV.

Die Qualle Casstopea xamachana nimmt im Hungerzustand an Kirpergriébe
ab. Diese Abnahme ist bedeutender bei regenerierenden Individuen, und mit
der GréBe des zu regenerierenden Gewebsbetrages wiichst die Geschwindigkeit
der GréBenabnahme des Individuums. Das neue regenerierende Gewebe wiichst

32 Charles R. Stockard, Studies of Tissue Growth. IV.

mit groBer Energie nach MaSgabe seiner auBerordentlichen Fihigkeit zur Nah-
rungseinverleibung auf Kosten der alten Kérpergewebe, und dieser Umstand ist
es, welcher die GriBenabnahme, das Schwiicherwerden und Abmagern des Kir-
pers veranlaBt. Eine weitgehende Abnlichkeit mit einer derartigen Wirkung
beobachtet man in den Fillen von gewissen malignen Tumoren.

Ubersetzt Gebhardt.

Literature cited,

Beess, S. P., ‘04, The Chemistry of Malignant Growths. II. The Inorganic
Constituents of Tumors. Am. Journ. Physiol. XII. pp. 167—172.

Emme, V., /06, The Relation of Regeneration to the Molting Process in the
Lobster. 36th Annual Report, Inland Fisheries, R. I.

Logs, J., ‘04, Uber den Einflu8 der Hydroxyl- und Wasserstoffionen auf die
Regeneration und das Wachstum der Turbellarien. Arch. f. d. ges. Physiol.
101. H.7/8. S. 340—348.

—— '05, Studies in General Physiology. Univ. Chicago Press.

MoraGan, T. H., '06, The Physiology of Regeneration. Journ. Exp. Zool. III.
pp. 459—500. -

STocKARD, C. R., ’08, Studies of Tissue Growth. I. An Experimental Study of
the Rate of Regeneration in Cassiopea xamachana. Carnegie Institution.
Pub. 103, and Science. N.S. XXVII. p. 448.

—— ‘09, Studies of Tissue Growth. IJ. Functional Activity, Form Regulation,
Level of the Cut, and Degree of Injury as Factors in Determining the Rate
of Regeneration. The Reaction of Regenerating Tissue in the Old Body.
Journ. Exp. Zool. VI. pp. 433—469.

ZELENY, C., ‘05, The Relation of the Degree of Injury to the Rate of Regen-
eration. Journ. Exp. Zool. HU. pp. 347—369.

Sonderdruck
aus Archiv fiir vergleichende Ophthalmologie Bd. I, H. 4, Seite 473—480.

The experimental Production
of various Eye Abnormalities and an Analysis
of the Development of the primary Parts of the Eye.

By
Charles R. Stockard,

Cornell University Medical School, New York City, U.S. A.

With two figures in the text.

While studying the influence of various substances on development
the writer found that it was possible to produce at will a number of
ophthalmic defects by the use of Mg, alcohol, cholreton, ether and other
anesthetics. The action of these substances seems to weaken or distroy
the dynamic processes necessary for the optic vesicles to push out from

Fig. 1. Anterior views of fishes heads showing different eye conditions produced by treatment with
Mg solutions.

the brain or to subsequently grow out to their lateral positions at the
sides of the head. In consequence of this, various degrees of the
cyclopean condition often occur among the fish embryos with which I have
experimented.

The cyclopean fish embryos are in all respects exactly comparable
to the human cyclops. One eye exists in the middle of the face an the
nasal pits are often represented by a single or double pit in front of
the eye. The eye conditions, as illustrated in the diagram Fig. 1, shwo
all steps in a series beginning with two eyes unusually close together,

174. Stockard: The experimental Production of various Eye Abnormalities.

VI, two approximated eyes, V, a double eye with two lenses, two pupils,
etc., IV, a laterally broad eye with a double retinal arrangement, and a
single lens and pupil and a typically single eye showing no indications

~
See
oe oe at, S
ba en sainy

Fig. 2. Young fish, a normal individual above and cyclopean monsters below.

of its double nature, II]. The later condition may be termed typical
or perfect cyclopia, from this we pass to extreme cyclopean eyes which
are unusually small, I, sometimes deeply buried in the head, others
with small optic cups and illfitting lenses which protrude beyond the eye,

Stockard: The experimental Production of various Eye Abnormalities. 475

and, finally, all retinal or optic cup portions of the eye may be absent,
with independent lenses present, or both optic cup and lens may fail to
form and eyeless creatures result, I. Many illustrations of all these stages
have been found and studies among the hundreds of cyclopean fish which
have been produced by these methods.

Some of the embryos present perfectly normal bilateral brains and
show no abnormality other than the cyclopean eye and characteristic
probocis-like mouth. The cyclopean eye occupies an antero-ventral posi-
tion, Fig. 2, and many fish with such an eye hatch from the egg and
swim about for a month or more in a perfectly normal fashion, the cy-
clopean eye functioning as an effecient organ of sight.

The development of the cyclopean eye in human monsters has been
difficult to interpret on account of the scarcity of material and want of
early stages of the defect. Such abnormalities are not readily explained
from later stages. In the summer of 1906, when these monstrous fish
were first produced, I secured only later stages and on finding all degrees
of union between the eyes concluded that the cyclopean condition resulted
from a more or less intimate fusion of the two eye components after
they had arisen from the brain. This position has been held by other
workers both before and since my study. A more careful investigation,
however, of the earliest stages of cyclopea in the living eggs and in
sections shows that the final condition of the eye is foreshadowed in the
first appearance of the optic anlage from the brain. The early eye is
either perfectly single or duble from the start, and the union of the two
components does not become more intimate during development, even
though the eye may develop partially within the brain itself.

In addition to the above series showing the various degrees of cy-
clopia, another series of ophthalmic defects were induced by the same
chemical substances. These individuals have one perfectly normal eye
in its usual position on either the right or left side of the head while
the eye of the other side may be either smaller than usual, or very
small and defective in structure, or deeply buried in the head tissues as
a choroid vesicle entirely failing to form a typical eye, or finally, the
optic vesicle of one side may not arise from the brain and thus the
individual has a perfect eye on one side and no indication of an eye on
the other. I have called such monsters as these ,.Monstrum Monophthal-
micum Asymmetricum“ as distinguished from the cylopean monster with
its one symmetrically placed eye.

This type of monster seems rarer in nature than the well known cy-
clops, although they do occur. I have known a child with one perfect

476 Stockard: The experimental Production of various Eye Abnormalities.

eye and the other small and defective, and Professor T. H. Morgan has
shown me a pigeon that presented a similar condition.

The asymmetrical monophthalmic monsters are possibly due to the
differing degrees of resistance to the anesthetics possessed by the anlagen
of the two optic vesicles. The doses used are of course very delicately
adjusted. One eye begins to push out from the brain slightly before the
other (in studying normal embryos it is often found that one organ of
a bilateral pair forms and develops ahead of the other for a time) the
weaker or slower eye is affected by the solutions while the stronger is
not. The different degrees of abnormality shown by one eye may be an
index to the more or less wide difference in developmental energy possessed
by the two eyes of the same individual.

It is of interest to record here that in some cases the development
of the auditory vesicle or ear is affected by the alcoholic solutions. At
times one ear is injured and the other not, in such individuals when
the eyes also exhibit an asymmetrical condition it invariably happens
that the defective eye is on the side with the defective ear. The rates
or strengths of development of the sides of the embryo are different, in
other words development is not perfectly bilateral, one side may go
ahead of the other for a time then the other catches up and may actually
ect ahead and so on.

Still other cases occurred in which both eyes were small and de-
fective although they pushed out from the brain and reached the sides
of the head. The two eyes of these mdividuals show the various degrees
of imperfection found in the small eye of the asymmetrical monophthalmic
monsters.

Alcohol was most effective of all the anzesthetics employed in pro-
ucing these conditions. Proper strength solutions of alcohol may cause
as many as 98 per cent of the eggs to develop into young fish exhibiting
the various ophthalmic defects mentioned above. Magnesium salts in
solution were next in efficiency, In one experiment 66 per cent of the
embryos were cyclopean or asymmetrically monophthalmic. The fish
were in better general condition in the Mg solutions than in other sub-
stances. Mg effects the development of the eyes without causing, in
many cases, defects or weaknesses in other parts of the central nervous
system. Alcohol on the other hand disturbs the development of the
central nervous system as a whole so that embryos are rarely strong
after treatment with it.

These experiments prove that many of the eye malformations met
with in nature are probably due to some abnormal condition in the deve-

-
A zs

Stockard: The experimental Produktion of various Eye Abnormalities. 477

lopmental environment having acted upon the early embryo. Anesthesia
tends to weaken or lower the dynamic processes of development and it is
probable that other causes which would interfere with normal nutrition
might cause similar effects. This would apply especially to the mammal-
ian egg where the yolk has been lost and the embryo depends upon a
perfect placentation for its proper nourishment from the mother. I suggest,
therefore, that cyclopea and other ophthalmic defects in mammals are
due to poor placentation or a diseased condition of the mother which sub-
jects the embryo to an abnormal environment during development. In
man such defects are probably often due to an alcoholic mother. There
is no evidence to indicate that these defects are the result of a peculiar
or abnormal germ cell, and against such a view the experimenter has
the power to cause at will perfectly normal eggs to develop into cyclopean
monsters by the use of alcohol and other anesthetic agents.

In a recent paper in the American Jour. of Anatomy, X, p. 369,
I have discussed the anatomical conditions of cyclopia and shall not men-
tion them here.

Development of the Primary Parts of the Eye.

The embryos discussed above furnish excellent material for a study
of the. relationship between the development of the optic vesicle and the
crystalline lens; the two primary parts of the eye which arise in the
embryo from different sources, the vesicle from the brain wall and the
lens from the head ectoderm. It has been claimed by several experimen-
ters that the optic-vesicle was entirely independent of the lens in its
development, while on the other hand, the lens was entirely dependent
upon the optic vesicle for its origin from the ectoderm as well as for
its later differentiation into the clear refractive lens of the eye.

A study of embryos having no optic vesicles, others with a vesicle
on only one side and finally those with a median cyclopean eye show
the following facts regarding the relationship between the development
of the optic vesicle and optic lens.

The crystalline lens may originate from ectoderm without any direct
stimulus whatever from an optic vesicle or cup. These self-originating
lenses arise from regions of ectoderm that are not in contact with either optic
vesicle, the brain wall, or any nervous or sensory organ of the individual.

The lens-bud is capable of perfect self-differentiation. No contact at
any time with an optic vosicle or cup is necessary. These lenses finally
become typical transparent refractive bodies exactly similar in histological
structure to a lens in the normal eye.

17S Stoekard: The experimental Production of various Kye Abnormalities.

The size and shape of the lens is not entirely controlled by the
associated optic cup. Lenses may be abnormally small for the size of
the cup, or entirely too large, so that they protrude; or, finally, peculiarly
shaped oval or centrally constricted lenses may occur in more or less
ordinarily shaped optic cups. The lens is by no means always adjusted
to the structure of the optic cup as has been claimed by some observers.

An optic vesicle or cup is invariably capable at some stage of its
development of stimulating the formation of a lens from the ectoderm
with which it comes in contact. It is remarkable how extremely small
an amount of optic tissue is capable of stimulating lens formation from
the ectoderm.

The optic vesicle may stimulate a lens to form from regions of the
ectoderm other than that wich usually forms a lens. This is shown by
the fact that a median cyclopean eye always stimulates a lens to form
from the overlying ectoderm. It is scarcely possible that the lateral
normal lens-forming ectoderm could follow the cyclopean optic cup to
the many strange situations it finally reaches.

The ectoderm of the head region is more disposed to the formation
of lenses than that of other parts of the body, since free lenses invariably
occur in this region.

A deeply buried optic vesicle or cup may fail to come in contact
with the ectoderm; in such cases it lacks a lens. The tissues of the
embryonic cup in the fish are unable to form or regenerate a lens. This
is not true for all embryos as has been shown for one species of frog.

The optic lens may be looked upon as a once independent organ
(possibly sensory or perhaps an organ for focusing light on the brain
wall, before the vertebrate eye had arisen) which has become so closely
associated with the nervous elements of the eye that it has to some extent
lost. its tendancy to arise independently, although still capable of doing
so under certain conditions. The lens now arises much more readily in
response to a stimulus from the optic vesicle, a correlated adjustment
which insures the almost perfect normal accord between the optic cup
and the lens.

Finally it may be stated in brief, that the optic vesicle or cup
always has the power to stimulate a lens to arise from any ectoderm
with which it may come in contact during certain stages of its development.
Secondly, the ectoderm of the head region also has the power under
proper conditions to form an independent lens which will differentiate
perfectly without the stimulus or presence of an optic vesicle or cup. -

Stockard: Die experimentelle Erzeugung verschiedener Abnormititen des Auges. 479

Kurze deutsche Inhaltsangabe zu vorstehender Arbeit:
Die
experimentelle Erzeugung verschiedenartiger Abnormitaten

des Auges nebst einer Erorterung Uber die Entwicklung der
Hauptteile des Auges. i

Stockard studierte den Einflu{8 des Magnesiums, Alkohols, Chlor-
iithyls, Athers und anderer Anisthetika auf die Entwicklung des Fischauges.
Er erhielt so u. a. verschiedene Grade cyklopischer Mibbildungen, die
in allen Hinsichten der menschlichen Cyklopie vergleichbar waren. Auf
der einen Seite der Reihe stehen die Falle, wo zwei getrennte Augen,
die nur naher als normal beieinander liegen, vorhanden sind, den Uber-
gang bildet ein median gelegenes, duberlich einfaches Auge mit doppelter
Retina, der héchste Grad wird durch eimen medianen hochgradigen
Mikrophthalmus oder durch vélligen Mangel der Augenanlage dargestellt.
Die Wirkung der erwahnten Chemikalien ist also eine hemmende. Es
zeigte sich tibrigens bei friihen Stadien, dai die Cyklopie keinen Ver-
schmelzungsprozebh vorher isolierter Anlagen darstellt, sondern dali solche
Augen von vornherein einfach oder doppelt angelegt sind.

Eine andere seltenere Art von Mifbildungen ist durch Vorhandensein
eines normal gelagerten, gut entwickelten und eines symmetrisch gelegenen
unvollkommen entwickelten Auges charakterisiert (Monstrum monophthal-
micum asymmetricum). Es ist anzunehmen, dab in diesen Fallen die
eine Augenanlage weniger widerstandsfahig ist oder etwas spater aus-
wichst als die andere und dali die Giftwirkung gerade hinreicht, diese
letztere zu schadigen.

Zuweilen trifft auch mit einseitiger Mibbildung des Auges eine eben-
solche des Ohrapparates zusammen, dann finden sich beide Storungen auf
der gleichen Kérperseite, was obige Auffassung von der verschiedenen
Qualitaét der Kopfhalften bestatigt.

Andere Fille zeigen zwei richtig gelegene, in verschiedenem Grade
mifbgebildete Augen.

Starker Alkohol rief iiber 98°/, Mibbildungen hervor; Magnesium
in einem Falle 66°/, bei besserem Allgemeinzustande des Tieres, wihrend
beim Alkohol die Schaidigung des Zentralnervensystems gréfer ist, so dab
die Tiere selten kraftig sind.

Diese Versuche zeigen, dali viele Augenmifbildungen von ungeeigneter
Ernahrung des Embryos herriihren kénnen (z. B. Alkoholismus der Mutter),
wahrend kein Beweis fiir eine abnorme Beschaffenheit der Keimzelle vor-
handen ist.

{8Q Stockard: Die experimentelle Erzeugung verschiedener Abnormititen des Auges.

Entwicklung der Hauptteile des Auges.

Besonderes Interesse bietet das gegenseitige Verhalten von Augen-
becher (bzw. -blase) und Linsenanlage.

Im Gegensatze zu bisherigen Annahmen zeigte sich, dab das Kopf-
ektoderm ganz unabhangig von dem Vorhandensein einer Augenblase
imstande ist, eine vollig entwickelte Linse zu liefern, auch beztiglich der
Dimensionen besteht keine feste Abhingigkeit der Linse vom Augen-
becher.

Jede Augenanlage hat die Fahigkeit, das Ektoderm zur Linsen-
bildung anzuregen, auch diejenigen Teile derselben, die gewéhnlich keine
Linse produzieren. Wenn die Augenanlage tief im Kopfe liegt und das
Ektoderm nicht erreicht, so fehlt die Linsenanlage.

Die Linse kann als ein Organ angesehen werden, das einmal selb-
stiindig war und diese Selbstandigkeit nur ausnahmsweise einmal wieder-
erlangt, wahrend es jetzt gewdhnlich in Abhangigkeit von den nervésen
Teilen des Auges ist. G. Freytag (Miinchen).

Reprinted from THe AMERICAN JOURNAL OF ANATOMY, VOL. 11, No. 2
January, 1911

THE ANATOMY OF THE THYROID GLAND OF ELAS-
MOBRANCHS, WITH REMARKS UPON THE HYPO-
BRANCHIAL CIRCULATION IN THESE FISHES

JEREMIAH S. FERGUSON
Assistant Professor of Histology, Cornell University Medical College

TWENTY FIGURES

NERVOUS Gs tute ng ne eae ne oe ee ee ee ee 151
peat ma IRM PRANTL oe aoe oo aig ook ald Lois 2 0, 4c dsb «me RGR ein eka boa ca ecden 153
SMe REeDUNRIRPRERL EINES Foc fo coe orcs ae wrn ws ss os a a. 0.2 nen 1b ho bank capeveccis 164
The anatomical relations of the thyroid gland........................... 165
mre eerie tees Mion 22h Jae i vc Lk ee Sad) A eels 171
The hypobranchial circulation and the origin of the thyroid vessels....... 175
Vomeand lympnatics of the thyroid region.............-..-2--..20c0<0.+0:-. 183
The histology of the Elasmobranch thyroid gland........................... 187
OTE PIT sy heed och OEMS Sikes en rr er ey 207
retire saat ee ee 9s. ie LA... cs cae ess oe sd ve Sa Le poate We 209
aI I RR ok cin oe wo 00 5 ono tase ap 4 mat Sv eres 210
INTRODUCTION

Even a casual reference to the literature of the thyroid gland is
sufficient to indicate that the organ has been more carefully stud-
ied in most all other classes of animals than in the Elasmobranchs.

The organ may be said to make its first appearance in the As-
cidians, Amphioxus, and Cyclostomesasa depressed groove, trough,
or series of recesses in the ventral floor of the pharynx, usually
known as the endostyle, or the hypobranchial or hypopharyn-
geal groove, which, as first shown by W. Miiller (’71) who studied
Myxine glutinosa and Petromyzon, is to be considered the homo-
logue of the median thyroid of the vertebrates. In the Cyclo-
stomes the structure, relations and development of the primitive
thyroid have been more recently studied by Guiard (’96), Cole
(05), Schaffer (’06), and Stockard (’06). The structure of the
organ is very simple and only partially resembles the thyroid of

152 JEREMIAH S. FERGUSON

higher vertebrates. Possibly the thick tenacious secretion formed
by the endostyle, upon the presence of which the function of the
organ very largely depends, may well be taken to bear a relation
to the colloid material which is so characteristic of the mammalian
gland. Inasmuch as the retention of an albuminous secretion
within the glandular lumina of the animal body, a condition
frequently observed by the pathologists and normally present in
other glands as well as the thyroid, e.g., mammary gland, kidney,
hypophysis cerebri and parathyroid gland, leads to the accumu-
lation of a colloid material bearing a more or less striking resem-
blance to the colloid material in the follicles of the thyroid gland,
the deduction from the phylogenetic standpoint, that the reten-
tion within the follicles of the thyroid of a once free mucous se-
cretion would account for the colloid character of the follicular con-
tent, would not seem inappropriate. The character of cells
which pour forth the free secretion of the endostyle or hypobran-
chial thyroid of the Ascidians, Amphioxus and Cyclostomes is not
so very different from the colloid secreting cells of the thyroid
follicles of mammals.

In the Teleostei, Wagner (’53) has studied the form and loca-
tion of the thyroid gland and directed attention to the similarity
of its structure to that of mammals. Simon (’44) and Baber (’81)
have given extended descriptions of the thyroid gland in several
species of bony fishes; Maurer (’86) described the structure and
studied fully the development of the thyroid gland of the carp
and trout. In the more recent literature the structure of the thy-
roid in fishes seems not to have received the attention which it
apparently deserves.

Extended descriptions of the thyroid gland of reptiles are
found in the articles by Simon (’44) and Baber (’81). De Meuron
(86) studied the organ in Lacerta. Van Bemmelén (’87) described
the gland in Hatteria and Lacerta as being transversely placed
over the trachea near the heart, and as forming a small, thin
unpaired organ. Guiard (’96) has also discussed the structure of
the organ in reptiles.

In the Amphibia the work of Maurer (’88) and the excellent
descriptions by Wiedersheim (’04) apparently leave little to be

THE ANATOMY OF THE THYROID GLAND 153

desired, though the organ has been much studied in this class of
animals.

In Aves the thyroid gland has been extensively studied by
Simon (’44), Peremischko (’67) who considered the histology
as well as the gross anatomy of the organ, Baber (’81) and De
Meuron (’86). The literature of the structure and development
of the thyroid in the chick is extensive.

In most of the Mammalia the anatomy of the thyroid gland is
well known and its literature has acquired voluminous proportions.
Its review does not fall within the scope of the present paper.

REVIEW OF THE LITERATURE

A careful study of the available literature has revealed, with the
exception of the work of Guiard, only casual references to the
anatomy of the thyroid gland in the Elasmobranchs. Simon
(44) studied the Selachian (Squalus) and the skate (Raia). He
describes the thyroid gland as ‘‘a single organ, situated in the me-
dian line, in connection with the anterior surface of the cartilages
which bind together the branchial arches of opposite sides of the
body,” and he states that it may lie in contact with the “lingual
bone,’ or may be more or less distant from the mouth, but that
it is ‘“‘always at the spot where the great trunk of the branchial
aorta distributes its terminal branches. It lies at the angle of
this bifurcation . . . ; it is covered by the sterno-hyoid
or sterno-maxillary muscle, and also by the myo-hyoid and genio- -
hyoid, when these are present.’’ His description I find to hold
good for Raia, but it does not entirely correspond to the position
of the thyroid gland of Squalus, Mustelus, or Carcharias. As to
its vascularization, Simon states that the gland receives its blood
supply by means of a recurrent branch given off by the first
branchial vein, while yet within the gill, and that ‘‘it never
receives the smallest share of supply from the branchial artery
with which it is in contact.’”’ The last portion of this statement
is precisely correct for all the species which I have examined,
though apparently at variance with the observations of some
authors: the first portion, as to the origin of the thyroid artery,

154 JEREMIAH S. FERGUSON

would appear to be not very accurately expressed, for it never
arises from the afferent branchial vessel which leaves the dorsal
end of the branchial arch, but, on the contrary, arises from the
ventral end of the nutrient or efferent loop. The relation of the
thyroid gland to the bifurcation of the ventral aorta is so intimate
as to readily suggest the error of other observers who have pre-
sumed that the organ received some blood directly from the first
pair of branchial arteries. Moreover, the thyroid artery passing
from its origin lies directly under, and in contact with the first
pair of afferent branchial arteries so that until these latter vessels
have been carefully dissected out of their sheath it is impossible
to determine with certainty that they have no connection with the
thyroid vessels. In the skate the gland lies directly upon the aor-
tic bifurcation and the pulsating blood-vessels are readily seen
through the transparent organ as soon as it is exposed.

Miiller (71) speaks of the thyroid gland of Raia clavata as a
flattened brownish-red body, lying at the point of division of the
branchial artery. It possesses a connective tissue capsule with
trabecula which divide the organ into a small number of lobes,
within which the connective tissue penetrates between the lobules.
The follicles possess a thin membrana propria and a cylindrical
epithelium; they contain a homogeneous, gelatinous yellowish
mass. The epithelium possesses a shiny cuticular border and
appears to send processes into the lumen. The description given
by Miiller holds good for Raia, the genus which he studied, but
it does not correspond to the condition of the thyroid gland of
Mustelus, Squalus or Carcharias, the difference being chiefly due
to the fact that in the Batoidei the connective tissue forming the
thyroid trabecula and interfollicular septa is apparently much
more abundant than in the Selachii.

Balfour (’78) discusses very briefly the early development of the
thyroid gland of Elasmobranchs prior to the appearance of a
lumen within its follicles. He does not consider the anatomy of the
organ in the adult.

Baber (’81) says that “‘in the skate the gland is single (with the
exception of a few detached vesicles) and forms a yellow, flattened,
lobulated body, occupying the median line at the bifurcation of

THE ANATOMY OF THE THYROID GLAND 155

the branchial artery. Anteriorly it sometimes presents a narrow
process of gland-tissue running forward, and behind it is limited
by the bifurcation of the branchial artery.”” The contents of its
vesicles consisted of coarsely granular masses or globules of various
sizes which ‘‘correspond with the ‘colloid substance’ of authors.”’
He surmises the non-existence of lymphatic vessels and says that
in both the skate and the conger-eel ‘“‘an extensive system of
vessels lined with epithelium becomes injected by the method of
puncture” ; he considers that these are blood-vessels. The narrow
process of glandular tissue which Baber says is occasionally
present in Raia is more frequently seen in the Selachii; it is con-
stantly present in all the examples of Carcharias which I have
dissected. It extends forward until it comes into contact with
the anterior margin of the basi-hyal cartilage (lingual bone) which
presents a depression, frequently amounting to a complete fora-
men, for the reception of the anterior extremity of the glandular
process with the connective tissue by which it is heavily invested.
This process is obviously analogous to the pyramidal lobe of the
mammalian thyroid, and as it extends all the way to the pharyn-
geal submucosa in many instances it may well be considered as
indicative of a phylogenetic connection of the gland with the cavity
of the pharynx, a condition which is also indicated by the on-
togeny of the organ in all the orders, and which appears to be
permanent in the Ascidians, Amphioxus, and the Cyclostomes.
Baber’s suspicion of the non-existence of lymphatics within
the thyroid gland appeared to the writer to be such a remarkable
observation and so out of harmony with the known anatomy and
physiology of the organ in the higher animal orders as to require
further study. This study was pursued by means of a consider-
able series of careful dissections with many injection experiments
and did not appear to confirm Baber’s opinion. Baber’s obser-
vation that the blood-vessels in these animals could be injected
by the method of puncture is quite accurate, but it does not by
any means disprove the existence of lymphatics. His results were
apparently dependent upon the fact that the smallest veins and
capillaries are of very considerable caliber and readily admit of
injection, while the lymphatics are very minute and are entered

156 JEREMIAH S. FERGUSON

only with difficulty and not frequently when the needle is thrust
into the substance of the gland.

Balfour (81) referring to the development of the organ in Scyl-
lium and Torpedo says that at first it is solid and attached to the
esophagus. “‘Eventually its connection with the throat becomes
lost, and the lobules develop a lumen.”’

Dohrn (’84) in his plate XI, fig. 5, indicates by outline the thy-
roid gland of Ammocetes, but does not illustrate or describe the
thyroid gland of the Selachii, though in his text he includes an
extended -description of the thymus of the latter animals. His
outline of the thyroid of Ammocetes does not conform to that the
the gland in the Selachii.

De Meuron (’86) says that in Scyllium the thyroid is elongated,
in Galeus and Acanthias, much flattened, in Raia pyramidal or
rounded. It lies just above the terminal bifurcation of the bran-
chial artery. . In Myelobates it les behind the os hyoideus,
beneath the sterno-mandibularis muscle, and is triangular in
shape, short, flattened, transversely elongated, and has a length of
2em. In Acanthias the thyroid gland presents an irregular con-
tour, certain groups of follicles being even completely detached,
and placed around the principal group. The observations of De
Meuron would appear to be accurate as far as they go but are
possibly founded upon the examination of too few individuals.
Thus in Squalus acanthias I found the thyroid frequently broken
as described by De Meuron for Acanthias but other individuals
presented a thyroid which was perfect, not the least broken up
or irregular in contour, and in the closely related Mustelus canis
irregularity of contour is certainly the exception, not the rule. In
Scyllium he says the thyroid is elongated and I find that superfi-
cial examination of the related species Carcharias, would indicate a
similar condition, but if the semi-opaque white mass of connective
tissue, in which the thyroid gland of Carcharias is heavily clothed
and so closely invested that it seems to form paart of the gland,
be dissected out and held, stretched in its normal form, between
the bright sun and the eye of the observer there is readily seen
within the reddish-white connective tissue mass the outline of the
yellowish-orange thyroid gland, which instead of having theelon-

THE ANATOMY OF THE THYROID GLAND 157

gated form of the outward mass is flattened, transversely elon-
gated, and of the same peculiar triangular or shield-like shape
which is characteristic of the organ in the dogfish and closely
simulated by that of Raia. It seems possible that the elongated
gland observed by De Meuron in Scyllium might be susceptible
to a similar analysis.

Guiard (’96) studied six species of the Selachii and five of the
Batiodei. In Scyllium he found the thyroid gland of pyriform
shape, the anterior extremity being prolonged forward as far as the
anterior margin of the lingual cartilage (“‘copule’’), where it passes
between the two lateral halves of the coraco-hyoid muscle. This
description, as given by Guiard, corresponds with the position and
form of the gland which I find in Carcharias and which, as regards
the anterior prolongation, appears to be analogous to the pyrami-
dal process of mammals. But Guiard’s fig. 1, in the absence of
specific contradiction in his text, might be taken to indicate that
the thyroid gland had been found beneath the coraco-hyoid
muscle; this is not the case in any of the species which I have
examined and I presume it is not the case in Scyllium catulus, from
which species his figure was drawn. In each species I have found
the gland lying, without exception on the ventral surface of the
eoraco-hyoideus, between it and the coraco-mandibularis, except
that at the anterior portion the gland lies between the coraco-
hyoid muscles of the two sides, the divergence of the two muscles
exposing the ventral surface of the cartilage at this point. In the
Batoideithe coraco-hyoidei are so widely separated that the whole
thyroid gland may come to lie directly upon the basi-hyal cartilage,
the aortic bifurcation and the coraco-branchialis muscles,
which are successively exposed from before backwards by the
separation of the coraco-hyoids, but in this case the fascia which
covers the ventral surface of the coraco-hyoids dips beneath the
dorsal surface of the thyroid gland.

In Acanthias vulgaris and Mustelus leevis Guiard as did De
Meuron, notes the tendency of the thyroid to present detached
vesicles, its contour being very irregular. In Galeus canis the
thyroid gland lies rather farther forward and is partially cevered
by a fold of the buccal mucosa. In Carcharias glaucus the

158 JEREMIAH S. FERGUSON

coraco-mandibularis muscle is relatively very broad and com-
pletely hides the thyroid gland; the gland is described as reniform
and voluminous. With the exceptions recorded the position of
the thyroid in the various species of Selachii corresponds fairly
well with that described for Scyllium.

Of the Batoidei, in Raia alba the coraco-hyoidei are small and
widely separated, and between these muscles the first pair of
branchial arteries emerge. The thyroid gland is described as lying
between the bifurcation of the aorta and the hyoid arch; itisa
very large globular organ and its deeper surface is slightly pro-
longed as far as the arterial bifurcation. In Raia oxyrhynchus
the thyroid gland in transversely elongated. In Raia pastinaca
the coraco-hyoidei approach one another and the gland is longi-
tudinally elongated; in this particular it corresponds to the Sela-
chian type. In this last species it is a large pyriform organ with
its broad end in relation with the hyoid cartilage, andits point
extending nearly to the bifurcation of the branchial artery; at
its point the gland presents a prolongation ‘‘which descends
between the branchial sacs to a depth of about 0.5 cm.”’ I desire
to call attention to the fact that in Carcharias a posterior prolonga-
tion appa:ently also exists and is constantly present, but so far as
I am able to observe it consists solely of connective tissue and con-
tains no glandular substance; it can not, therefore, be in any way
analogous to the anterior prolongation of the processus pyrami-
dalis. I think it is to be connected with the fascia of the thyroid
sinus, which will be discussed later on, rather than with the gland
itself.

Guiard sums up his work on the morphology of the thyroid
gland in the rays by saying that the organ lies beneath the coraco-
mandibularis, between the coraco-hyoids, is always globous, of
more of less pyramidal form, and with a prolongation backward
to the bifurcation of the “branchial artery.’”’ This corresponds
very well with the condition which I find in Raia Erinacea except
that in this species, at least, the gland constantly overlies the bifur-
cation of the ventral aorta (branchial artery), and that it is always
somewhat flattened, its ventro-dorsal axis being shortened. The
organ is relatively thicker than in the Selachians because of the

THE ANATOMY OF THE THYROID GLAND 159

presence of an increased amount of connective tissue between its
vesicles.

On page 26 Guiard says that the thyroid gland “‘of fish” isalways
unpaired; it is quite obvious that this remark should apply only to
the Elasmobranchs, the only order of fishes which Guiard appears
to have studied.

Bridge (’04) passes the thyroid gland with the brief statement
that “‘in adult Elasmobranchs the thyroid is represented by a
moderately large compact organ, situated near the anterior end
of the ventral aorta.’’ Although he describes the gland as one of
the ‘“‘blood glands” in connection with the vascular system, he
does not mention, nor indicate in any way, the source of its blood
supply. The statement of its intimate relation with the aortic
bifurcation might well lead one to erroneously suspect a supply
from this source. In quite another place (page 332) he speaks of
‘a remarkable system of arteries for the supply of nutrient blood
to the gills and heart,’’ which takes origin from the ventral ends
of the loops about the gill slits, the commissural vessels forming
by their union the “median longitudinal hypobranchial artery
which lies beneath the ventral aorta.’”’ He fails to mention the
ultimate ramifications of this system of vessels or its relation to
the thyroid gland, falling into the same error in this particular
as T. J. Parker, from whose plates Bridge takes his figures, and
upon whose description he appears to have largely based his
text.

The literature upon the blood supply of the Elasmobranch
thyroid begins with Hyrtl (’58) who first described the hypo-
branchial arterial system in the Batoidei, if we except the very
incomplete description by Monro (1787). Hyrtl described the
thyroid artery as the “Ramus thyreoideus seu submentalis”’
which takes origin from the “‘vein’”’ of the second gill sac, and which
gives off muscular branches to that part of the oral mucosa which
lies between the inferior maxilla and the tongue bone as well as the
Glandula thyreoidea.’”’ Hyrtl did not at this time describe a
median hypobranchial artery, this vessel being represented in his
description by two anastomosing vessels on either side of the me-
dian line which arise from the second and third arches and which

160 JEREMIAH S. FERGUSON

pass backward to supply the anterior coronary vessels. Hyrtl
very clearly pointed out that the posterior coronary vessels
arise from the subclavian artery in the Batoidea and in 1872 he
showed that these vessels (posterior coronaries) were absent in the
Selachii, a point emphasized at considerable length some years
later by G: H. Parker and Davis (’99). In 1872 Hyrtl extended his
description of the hypobranchial arterial system to the Selachii.
He found the thyroid gland to be supplied by the “Arteria thy-
reo-maxillaris seu submentalis’”’ which supplied the floor of the
mouth and thyroid gland as in the Batoidei but which took origin
from the ‘‘veins’’ of the first gill sac, rather than from the second
as he had previously described for the Batoidei. He also described
the ‘Arteria cardio-cardiaca,’”’ called later the ‘‘commissural”
and “‘longitudinal commissural” (T. J. Parker) and the commis-
sural and ‘‘lateral hypobranchial” (G. H. Parker and Davis),
which fused in the median line to form a median vessel (median
hypobranchial) and from which the coronary vessels were derived.
At this time Hyrtl emphasized the absence of the posterior coro-
nary branches of the subclavian in the sharks and called attention to
an anastomosis from the subclavian forward to the median vessel
from which the coronary arteries arose. This anastomotic vessel
has since been called by T. J. Parker the ‘‘hypobranchial artery.”

Turner (’74) injected the conus arteriosus and studied the
course of the afferent and efferent branchial vessels; the course of
these vessels is now well known. He neither mentioned nor ex-
cluded any relation to the thyroid gland, apparently not recogniz-
ing this organ, nor did he work out the ultimate connections of
any of the smaller cervical vessels.

T. J. Parker (’80) dese:ibed the venous system of Raia nasuta
and called attention to ‘‘the extraordinary number of transverse
anastomoses it [the venous system of the skate] presents, the results
being to produce numerous‘ venous cireles,’ comparable to the circle
of Willis in the arteries of the mammalian brain, and the circulus
cephalicus in the arterial system of bony fishes. There is also
a direct passage from the sinus venosus and back again, in four
different ways, namely: (1) by the hepatic sinus; (2) by thean-
terior part of the cardinal vein and the cardinal sinus; (3) by the
whole length of the cardinal veins and their posterior anastomosis;

THE ANATOMY OF THE THYROID GLAND 161

(4) by the lateral veins and the prolongation of the cardinal
sinus into which they open.”

I would like to direct attention to the presence of similar venous
anastomoses in the Selachii as well as in the Batoidei. These
anastomoses in the cervical region are frequent and voluminous.
A complete circular anastomosis surroundsthe mouth close to the
maxillary and mandibular cartilages. Though notsoreadily seen in
the Selachii, it follows the same course as in the skate in which fish
it isvisible through the skin and oral mucosa; it ends in a maxillary
sinus at either angle of the mouth, which is connected with the
orbital sinusand with thejugularvein. Thehyoidsinusesare simi-
larly connected across the median line near the ventral surface,
two anastomotic vessels, the anterior the larger, connecting the
opposite sides. This anastomosis bears a most important relation
to the thyroid gland. The anterior vessel is so large as frequently
to almost envelop the gland as in a capsule, the vessel is sub-
divided by fibrous partitions, or consists, rather, of a mass or
series of vessels within a common sheath, and from its relation to
the thyroid gland, in its more or less dilated condition it is more
truly a sinus than a vein; it is conveniently designated the thyroid
sinus. It fills and empties with each movement of the mouth and
gills as water is forced through the branchial clefts, thus function-
ing with the aid of extrinsic muscles after the manner of a venous
heart. When the fish is examined out of the water the violent
movement of the gills so distends the sinus as often to wholly
obscure the thyroid gland by the volume of its contained blood.
It is almost impossible to reach the gland by dissection from the
ventral surface without cutting the sinus or some of its numerous
tributaries. The thyroid sinus receives the veins from the thyroid
gland, most of these vessels leaving the dorsal surface or posterior
margin of the organ.

T. J. Parker (’86) offers a description of the larger blood-vessels
of Mustelus antarcticus, which is, however, deficient as regards the
ultimate distribution of the smaller arteries. Exceptionsmay also
be taken to his statement of the distribution of the arteries which
constitute the rather remarkable hypobranchial arterial system,
which as already mentioned, bears an important relation to the thy-
roid gland. Parker’s description of the venous system is quite

162 JEREMIAH S. FERGUSON

accurate. The hyoid sinus is shown to empty into the jugular vein
with a valve at the orifice. The anastomosis between the hyoid
sinuses is considered, but its relation to the thyroid gland is not
discussed; since no mention of the thyroid gland is made it would
appear that this important organ was either overlooked or ignored.
Unless one is speciaily looking for the gland, in the effort to sepa-
rate the muscles without injury to the venous channels the organ
is easily broken up, and once disintegrated its particles are readily
lost amongst the mass of muscular tissue.

The tributaries of the hyoid sinuses are stated by Parker to
include the submental, posterior facial, internal jugular, and the
nutrient veins from the first hemibranch.

Parker’s description of the hypobranchial system follows that
of the ventral and dorsal aorta and begins with the subclavian
artery, which, he says, gives off the branchial and hypobranchial
arteries. Apparently he omits to mention the large lateral or
epigastric artery, whose course parallels that of the lateral vein,
though the beginning of the vessel is indicated but not namedin
some of hisfigures. Thehypobranchial artery described and figured
as a continuation of the subclavian, after giving off the antero-
lateral artery—which I find to be distributed to the pericardium
and adjacent muscles—unites with its fellow of the opposite side,
passes forward 2 em. in front of the conus arteriosus, and forms a
plexus from which are given off the coronary arteries posteriorly,
and anteriorly the median hypobranchial artery. Theplexus com-
municates laterally by two commissural arteries on either side
with the longitudinal commissural vessels uniting the ventral
ends of the efferent branchial loops. One gathers from the descrip-
tion that the course of the circulation is from the subclavian artery
through the hypobranchial to the efferent branchial loops, a
direction which may be thus tabulated:

branchial

hypobranchial
coronary (paired)
median hypobranchial (azygos hypobranchial)
commissural (two pairs)

antero-lateral (paired)

THE ANATOMY OF THE THYROID GLAND 163

Mention is not made of the lateral nor of all the coronary arteries.
Parker’s observations were made on Mustelus antarcticus. I
have dissected three species of the Selachii and one of the Batoidei,
I have not only been unable to confirm the course of the circulation
as indicated but I find that beyond the so-called hypobranchial
artery the course of the circulation is in the opposite direction, viz.,
from the efferent branchial loops to the coronary vessels and
systemic capillaries, and the hypobranchial artery serves as a
relatively unimportant anastomosis which, in these species is not
even constantly present. In addition to the pair of coronary
arteries distributed to the ventricle I have in my specimens
observed a dorsal artery which ramifies largely in the wall of
the auricle. The mandibular artery as described and figured by
Parker, is the one from which in my preparations the thyroid
artery is sometimes, though not constantly, derived. His descrip-
tion leaves one somewhat in doubt as to the origin of this vessel,
but he has figured it correctly as coming from the first efferent
branchial loop. His coraco-mandibular artery, derived from
the mandibular, is a vessel which apparently corresponds with that
which distributes its main branches, in my preparations, within
the thyroid gland and only incidentally gives small branches to
the coraco-mandibular and coraco-hyoid muscles; I have there-
fore called this vessel the thyroid artery.

G. H. Parker and Davis (’99) inan article on ‘‘ the blood-vessels
of the heart in Carcharias, Raia, and Amia’”’ repeated the work of
Hyrtl (’58 and ’72) so far as it immediately concerned the origin
of the coronary vessels, but being concerned only with the cardiac
vessels they made no mention of the thyroid artery or other deriva-
tives of the first hemibranch, nor of the gastric and pharyngeal
branches which arise in close relation to the anterior coronaries.
They described ‘‘the irregular longitudinal artery by which the
ventral ends of some or all of the efferent branchial arteries of a
given side are brought into communication,” hitherto referred to as
“longitudinal commissural”’ vessels (T. J. Parker) or as part of the
Arteria cardio-cardiaca (Hyrtl), and called the vessels the ‘‘lateral
hypobranchial artery,” reserving for the name commissural ‘‘ those
arteries which leave the lateral hypobranchials on their median

164 JEREMIAH S. FERGUSON

sides and, after more or less tortuous courses, unite with one
another in the median plane”’ to produce by their union the median
hypobranchial artery. The ventral continuation of the subclavian
artery they call the ‘‘coracoid artery.”’ Concerning the anasto-
mosis of this vessel with the median hypobranchial formed by the
hypobranchial artery of T. J. Parker they speak as follows:
‘‘\oreover neither of these vessels [median and lateral hypobran-
chial| can be properly considered a dependency of the subclavian,
for the branch which leaves that artery, and which T. J. Parker
regarded as their root, may be connected with them, as Hyrtl
(58, p. 17, Taf. 2) has shown, by only a relatively small vessel.
The union, then, is not in the nature of a continuous trunk, but an
anastomosis, and the vessel posterior to this union must be con-
sidered in the light of an independent artery. This we have called
the coracoid artery.”

MATERIAL AND METHODS

For the purposes of the present study I have dissected 32 speci-
mens of Mustelus canis, 10 of Carcharias litoralis, 3 of Squalus
acanthias, and 14 of Raia erinacea. In addition to these I have
had access to a number of sections from various Elasmobranchs
prepared by my late assistant, Dr. Guy D. Lombard. Themost
of these animals were dissected through the courtesy of The Wis-
tar Institute of Anatomy at the Marine Biological Laboratory at
Wood’s Hole, Massachusetts. My thanks are due these institu-
tions for the opportunity afforded.

The form and position of the thyroid gland was carefully observed
in each instance and its vascular connections determined bothby
dissection and by various methods of injection. The injections
were made chiefly with a hypodermic needle of very fine caliber,
though finely drawn-out glass tubes were used with somesuccess.
For pressure an aspirating syringe was used for routine work and
served very well; air pressure was also used at times. For tracing
the lymphatics, injections were frequently made into the substance
of the thyroid gland and into the connective tissue about the thy-
roid blood sinus and the other cervical blood vessels. For tracing

THE ANATOMY OF THE THYROID GLAND 165

the blood-vessels injections were made into both remote and near-
by vessels, the points selected including the hyoid and thyroid sin-
uses, the thyroid artery, the efferent branchial loops and commis-
sural arteries, the median hypobranchial artery, the coronar'y ar-
teries, the ventral aorta, conus arteriosus, cardiac ventricle and
auricle, the caudal artery and vein, the mesenteric artery and the
dorsal aorta. Injections from these various points were made not
only because of expediency in agiven species but for the special pur-
pose of determining the direction of flow and the relation of the
vessels to the thyroid circulation; hence, injections were made from
both sides of the branchial circulation, in the direction of the flow in
the veins and the arteries while other injections were made in a
direction opposite to the usual course of the circulation on the
arterial side, though this was, of course, impossible in the veins
because of the presence of valves.

Many of the thyroid glands were cleared and mounted in toto.
This was best accomplished with those from Mustelus, in which
species the gland is very thin. Some of the others were cut free-
hand into thick sections. These preparations gave very good
pictures of the lymphatics and blood-vessels except in the case of
the very thick glands. Still other thyroid glands were sectioned
for histological study.

THE ANATOMICAL RELATIONS OF THE THYROID GLAND!

The thyroid gland is more or less closely related to most of the
structures of the ventral cervical region, a region included between
the mandible in front, the coracoid arch or shoulder girdle behind,
and the branchial clefts on either side. This region forms the ven-

1The fact that the thyroid gland may be readily overlooked in the Selachii
is amply demonstrated by the frequency with which this region has been studied
and the almost entire absence of any adequate description of the gland. A brief
description of the methods of dissection which may be relied upon to locate and
expose the gland is offered in the hope that it may materially aid future investi-
gation of this organ.

The thyroid gland of Elasmobranchs can be readily reached from either the
oral or the cutaneous surface. By the cutaneous route two methods are especially
serviceable, the one by longitudinal, the other by transverse incision.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 11, No. 2

166 JEREMIAH S. FERGUSON

tral wallof the pharynx and contains the whole course of the ventral
aorta and its immediate branches. In Raia the heart is contained
within this area at its posterior margin, lying in the median line
just in front of the cartilaginous arch, but in Mustelus, Squalus
and Carcharias the heart has been pushed backward and lies
just beneath the coracoid arch.

The skin of the ventro-cervical region is thin but very tough;
laterally it is folded upon itself at the branchial clefts, on the inner
surface of which it becomes continuous with the mucosa covering
the gills. The fibers of the mylohyoid and geniohyoid muscles
are attached to the derma over the greater part of the ventral
cervical area. These muscles form a thin sheet, thickest and most
prominent in Carcharias, thinnest and frequently almost wanting

The first method is the more applicable in Raia, where the skin is loosely
attached and the coraco-mandibular muscle is small, thin and easily lifted. With
some variations I have followed the method outlined by Lombard (09). A
longitudinal incision is made in the median line through the skin and membranous
constrictor pharyngis. The skin and adherent muscle are dissected away and
retracted laterally, exposing the coraco-mandibularis. (Fig. 1B.’ A probe is
passed beneath the muscle which, after being well freed, is divided midway
between the mandible and the coracoid arch. The anterior flap is grasped and
reflected forward, at the same time dissecting away from its dorsal surface the
deep cervical fascia in which the thyroid gland is embedded. The gland is easily
recognized by its deep yellowish orange color and its peculiar rounded or triangu-
larform. In those exceptional instances when the thyroid gland is displaced for-
ward in Raia the anterior division of the coraco-mandibularis will have to be
reflected forward all the way to its mandibular insertion before the gland is
fully exposed; ordinarily the organ will be found directly over the aortic bifurca-
tion about midway between the mandible and the point at which the muscle was
bisected.

The above method is less easily applied to the Selachii for the reason that the
skin and the constrictor pharyngis are much more firmly adherent to the under-
lying structures than in Raia; moreover, in reflecting forward the anterior divi-
sion of the coraco-mandibularis one is almost certain to injure the thyroid sinus,
deluging the part with blood, before the gland can be exposed. In Carcharias
one encounters the added disadvantage that the thyroid gland is quite firmly
united to the coraco-hyoideus and the surface of the basi-hyal cartilage, and the
gland is buried in a mass of connective tissue by which it usually is entirely ob-
scured even after the coraco-mandibularis has been completely reflected away
from its surface. The second method is, therefore, the more applicable jin Mus-
telus and Squalus and is very much more certain in Carcharias. One blade of
a blunt scissors is inserted into the first branchial cleft on the right and then on
the left side and the clefts lengthened to their extreme ventral limits, the ends

THE ANATOMY OF THE THYROID GLAND 167

in Raia, and subject in all species to great individual variation in
volume. The muscles take origin posteriorly from the coracoid
arch, anteriorly from the hyoid arch and mandible, and laterally
from the outer surfaces of the branchial arches. Acting from
these ‘‘fixed points’ upon the more movable, but tough and in-
elastic skin, these muscles form a very powerful constrictor of the
pharynx and collectively are very properly termed the “‘con-
strictor pharyngis” (fig. 1, A). In addition to this constriction
the muscular contraction at the same time tends to draw open the
branchial clefts, thus permitting the more ready passage of water
during the rhythmic pharyngeal contraction or respiratory move-
ment. The muscular fibers of the constrictor pharyngis are inti-
mately adherent to the derma.

of the incisions exposing the margins of the coraco-hyoideus muscle. One blade
of the scissors is then pushed beneath the skin whereit readily passes between
the coraco-hyoideus and coraco-mandibularis muscles (fig. 1); the incision is
continued across the median line from side to side. This divides the coraco-man-
dibularis; its anterior portion is grasped with the forceps, lifted, and a longi-
tudinal incision through the skin and fascia carried forward along either margin
of the muscle. In the dogfish the divided muscle with the attached skin is easily
raised and the loosely attached deep cervical fascia dissected away from its
dorsal surface, exposing the thyroid gland. In Carcharias it is better to dissect
the deep cervical fascia away from the ventral surface of the coraco-hyoideus
muscle, rather than from the coraco-mandibularis; the thyroid gland is then
raised with the latter muscle and dissected out from the mass of connective tissue
which envelopes it. Finally, the gland must be dissected away from its anterior
attachment to themargin of the basi-hyal cartilage, or, in Carcharias, to a median
depression, in the ventral surface of this cartilage, which corresponds to the fora-
men caecum linguae of mammals; occasionally this depression is a true foramen,
in which case the thyroid process becomes obviously analogous to the lobulus
pyramidalis of themammalian thyroid gland. Thislobuleis represented in Muste-
lus by a short triangular projection, not constantly present, which overlies a
shallow median groove in the anterior margin of the cartilage. In Raia a similar
condition is much less frequently present.

The thyroid gland is readily accessible from the oral cavity. A needle passed
through the oral mucosa just in front of the basi-hyal cartilage—‘“‘lingual bone’’
—enters the substance of the thyroid gland if directed backward in Mustelus
and Squalus, well backward and close to the cartilaginous surface in Carcharias,
or backward and slightly ventralward in Raia. A transverse incision through
the oral mucosa, parallel to and just in front of the basi-hyal cartilage, exposes
the anterior margin of the thyroid gland and it may then be readily dissected
out from Mustelus or Raia, though with greater difficulty from Carcharias or
Squalus.

168 JEREMIAH S. FERGUSON

Removal of the integument with the adherent constrictor
muscles exposes the coraco-mandibularis (fig. 1, B), a slender
paired muscle, its two sides intimately fused in the median line,
which takes origin by a tendinous fascia from the ventral surface
and anterior margin of the coracoid arch. The paired muscle
passes ‘forward to its insertion, ending in short, rounded and
slightly divergent tendons which are attached to the posterior
margin of the inferior mandible. The muscle is inclosed within
the folds of a superficial cervical fascia, which forms its aponeu-
rosis and extends laterally to the surface of the branchial arches,
but on either side of the muscle, the aponeurosis fuses with the deep
cervical fascia with which it is in more or less close contact.

On lifting the coraco-mandibularis with its superficial cervical
fascia the coraco-hyoid muscle (fig. 1, C) is exposed; it is similar
in shape and appearance to the coraco-mandibular, but is much
broader, its lateral margin projecting from beneath the coraco-man-
dibularis, and in Mustelus, Squalus and Carcharias extending lat-
erally almost to the ventral ends of the branchial clefts, or even
overlapping them somewhat. In Raig the gills are more widely
separated, leaving a broad portion of the floor of the pharynx
exposed at the side ot the coraco-hyoideus in the anterior portion
of the cervical region. Posteriorly the several divisions of the
coraco-branchialis muscle cross this exposed portion of the pharyn-
geal floor to be inserted into the branchial arches.

The ventral surface of the coraco-hyoideus is smooth, its dorsal
surface separates into several muscular processes, the musculus
coraco-branchialis (M. ¢. br., fig. 5), to be inserted into the mem-
branous floor of the pharynx by a tendinous fascia overspreading
and firmly adherent to the fibrous pharyngeal submucosa and
the surfaces of the cartilaginous branchial arches. The divergent
portion of the coraco-hyoidei, on either side of the median line,
are similarly inserted into the movable basi-hyal cartilage (lingual
bone), so that the combined coraco-hyoid and coraco-branchial
muscles, arising from the anterior border of the coracoid arch, form
a very powerful dilator of the mouth and pharynx.

As the coraco-hyoideus does not extend forward beyond the
hyoid arch it exposes the membranous floor of the oro-pharynx

THE ANATOMY OF THE THYROID GLAND 169

Fig. 1. Dissection of the thyroid gland of Mustelus canis. In A the skin has
been reflected to show the superficial ‘‘constrictor pharyngis’’ muscle. In B
the ‘constrictor pharyngis’’ has been removed and the coraco-mandibularis
exposed. In C the coraco-mandibularis has been divided, exposing the thyroid
gland lying upon the coraco-hyoideus. (See page 209 for explanation of abbre-
viations used in all figures.)

170 JEREMIAH S. FERGUSON

between this point and the inferior mandible. The cartilages
of the hyoid arch except only the dorsal surface of the basi-hyal,
are loosely adherent to this membranous floor so that when
the mouth has been closed and the pharynx contracted the
tongue-like basi-hyal cartilage is pushed forward, producing a
deep fold in the oral mucosa. A needle thrust through this fold
in the median line, passing dorsal to the cartilage, penetrates
directly into the thyroid gland.

Not all of the ventral surface of the basi-hyal cartilage is
covered by the insertion of the coraco-hyoideus muscle; the por-
tion of the cartilage thus exposed varies in different species and
to some extent in individuals. In Carcharias and Raia the muscle
is inserted into only a small portion of the cartilaginous surface,
while in Mustelus all but a narrow anterior margin is covered by
the muscle fibers. The thyroid gland typically les upon this ex-
posed cartilaginous surface, extending backward for a greater or
less distance upon the ventral surface of the coraco-hyoideus
(fig. 1,C). In Mustelus and Squalus the ventral surface of the
basi-hyal cartilage has a raised arciform anterior margin with
a very slight medial depression, in Raia it is nearly flat, and in
all these species the thyroid gland overspreads the cartilage like
a thin membrane whose convex anterior border nearly corres-
ponds with the outline of the cartilage; in Carcharias the cartilage
presents a deep median groove or furrow into which the thyroid
gland sinks, lying there in a gelatinous mass of connective tissue
so voluminous that the gland is partially, sometimes wholly,
obscured.

In the dogfish and shark the thyroid gland is rarely pushed for-
ward beyond the margin of the basi-hyal cartilage; in Raia the
organ may extend farther forward so that it rests in part upon
the membranous floor of the oro-pharynx. In one of the skates I
found the gland carried so far forward that it lay wholly in front
of the cartilage. In Mustelus and Carcharias individual variations
are much less frequent than in Raia, but in Raia in the majority
of individuals the gland lies directly upon the bifurcation of the
ventral aorta (fig. 7).

THE ANATOMY OF THE THYROID GLAND 171
THE THYROID VESSELS

The anterior margin of the thyroid gland is firmly attached
by a dense fibrous fold of fascia to the antero-ventral margin of
the basi-hyal cartilage. Its ventral surface is in contact with
the coraco-mandibular muscle, to which it is firmly united by
a fascia. Its dorsal surface rests upon the basihyal cartilage and
the insertion of the coraco-hyoid muscle as described in the
preceding section. The lateral angles and posterior margin of
the thyroid gland are continuous with a fold of the deep cervical
fascia which is placed between the coraco-mandibular and coraco-
hyoid muscles, loosely attached to the opposed surfaces of these
muscles, but more firmly fixed to their lateral margins, so as to
form an aponeurosis for each. This fold of the fascia is much
thickened anteriorly where it approaches the margin of the thyroid
gland: at this point it incloses a transverse anastomosing vein
which connects laterally with the hyoid sinuses.

At the posterior margin of the gland the fascia splits, a thin
layer passing dorsally between the gland and the coraco-hyoid
muscle, a thicker portion extending forward over the ventral sur-
face of the organ, between it and the coraco-mandibular muscle.
This ventral division is of special importance; it contains the large
thyroid sinus consisting of an intricate net of veins and lymphatics,
which connects laterally with the hyoid sinus and in the median
line spreads over the ventral surface of the thyroid gland so that
when distended with blood it entirely obscures the organ.

The thyroid sinus is surrounded with connective tissue con-
taining a network of lymphatic vessels. Ink injected in the
living animal into the space between the lateral margin of the
thyroid sinus and the ventral end of the first branchial cleft
will after a few minutes be found filling many of the lymphatie
vessels of the thyroid sinus as well as many other perivascular lym-
phatics in relation with most of the cervical veins and the arteries
of thehypobranchial system (fig.8). Thelymphaties of the thyroid
plexus are sO mumerous and anastomose so freely that when
filled with ink they form a sac-like investment entirely obscuring
the sinus and the thyroid gland (fig.2). An attempt to inject with

172 JEREMIAH S. FERGUSON

a fine needle directly into the thyroid sinus may force the fluid into
either the venous or the lymphatic plexus, according as the one or
the other system of vessels happens to be entered. I am convinced,
as a result, of my injection experiments, that the lymphatics open
freely into the veins of this part, after the manner of the ‘‘ vasa lym-
phatica’’ of Favaro (vide infra). These observations are of interest
in connection with Baber’s inability to find lymphatics in the thy-
roid gland as indicating the relation between the vascular systems.
I find, however, that it is not possible for fluids injected into the
thyroid sinus or its plexus of lymphatics to pass in any quantity
‘into the vessels within the thyroid gland; this is presumably
because of the presence of valves.

The alternate expansion and contraction of the mouth and phar-
ynx, forcing the stream of water through the branchial clefts,
alternately fills‘and empties the thyroid sinus, so that by means of
these respiratory movements the sinus acts somewhat after the
manner of a venous heart. In this connection it is interesting
to consider the observation of Favaro (’06), as quoted by Sabin
(09), that the relation of the veins and lymphatics in fishes is
much more primitive than in mammals and that both lymph-
hearts and vein-hearts may be present in these animals. The
emptying and filling of the veins can readily be seen in the Selachi
or Raia on removing the skin, or even through the integument in
the living skate, the colored blood showing readily through the
vascular walls. The relation of the lymphatics to the blood-sinus
is so intimate that they must also beemptied and filled in the same
way, though since they contain a colorless fluid they can not be so
readily observed. I have, however, demonstrated that a colored
fluid injected into these lymphatic vessels will overspread the
thyroid region in ten to fifteen minutes and will almost entirely
disappear within the next fifteen minutes; the lymphatic circu-
lation must therefore proceed with considerable rapidity.

The thyroid artery approaches the organ from either side; in
Mustelus and Squalus it enters at the extreme lateral angle of the
triangular gland (fig. 19). In Raia, where the organ is of a more
rounded form it enters near the middle of the lateral border. The
branches of the artery ramify upon the surface of the organ send-

THE ANATOMY OF THE THYROID GLAND 173

| ul

WAC. hy.

Fig. 2. Showing the form and relations of the thyroid sinus when injected with
ink. A, in Raia. B, in Carcharias.

174 JEREMIAH S. FERGUSON

ing finer twigs into the interior. In this particular they offer an
interesting analogy to the condition found by Major (’09) in
man. Major says (page 484), ‘‘in the human the branching of the
large arteries takes place mostly upon the surface of the gland,
and having by their branching obtained their approximate dis-
tribution, ‘the smaller branches are sent in.” In the Elasmo-

‘Athyedestra 4 A.thyr sinestra----

Fig. 3. Outline of the arterial supply of the thyroid gland of Mustelus canis
as usually found. The vertical shading indicates the area supplied by the right
thyroid artery, the horizontal shading that supplied by the left.

Fig. 4. Showing the less usual distribution of the thyroid arteries; the shading
as in fig. 3.

branchs the condition might well be described in the same words;
this is the more remarkable inasmuch as Major states that this is
not the condition in other mammals, e.g., the dog and cat. The
Elasmobranchs, therefore, seem to harmonize with the human
rather than the lower mammalian condition as regards the dis-
tribution of the main branches of the thyroid arteries.

THE ANATOMY OF THE THYROID GLAND 175

The left thyroid artery usually supplies a greater portion of the
gland than the right (fig. 3), though the relative area is subject to
extreme variation and in occasional instances the ratio may be
reversed (fig. 4). The area of distribution in the great majority
of individuals is approximately as indicated in fig. 3.

THE HYPOBRANCHIAL CIRCULATION AND THE ORIGIN OF THE
THYROID VESSELS

In attempting to trace the circulation of the thyroid gland by
means of injection experiments I was at once struck with the diffi-
‘culty of reaching the gland by means of injections into the gill
arteries, the ventral aorta or the heart. It was obvious that the
thyroid artery has no direct connection with the ventral aorta or
the afferent branchial vessels, a fact which seems to have been first
observed by Simon (’44) who stated, without further explanation
or any outline of his reasons therefor, that the thyroid glandin Raia
‘never receives the smallest share of supply from the branchial
artery with which it is in contact.’”’ This fact seems to have been
seldom recognized and never sufficiently emphasized by later
writers.

The thyroid artery arises either from the mandibular artery, or
by a common trunk with this artery, from an arterial sinus at the
ventral extremity of the first branchial cleft (figs. 5 and 6); this
sinus forms the ventral portion or connecting vessel of the efferent
vascular loop contained in the hyoidean hemibranch and first holo-
branch. From the dorsal extremity of this same loop the first
efferent branchial artery passes to the dorsal aorta. It is there-
fore necessary for fluid injected into the ventral aorta or its imme-
diate branches to pass through the gill capillaries before it can enter
the thyroid arteries, and few injection fluids readily pass through
capillary vessels. On the other hand, fluid injected into the thy-
roid artery or, as I later found, into any portion of the hypobran-
chial system passes readily into the vessels of the thyroid gland;
such injections I repeatedly made, into the sinus at the ventral
end of the first efferent branchial loop, into the thyroid artery, the
median hypobranchial artery, and even into the coronary artery

176 JEREMIAH S. FERGUSON

taking care to prevent the escape of the fluid through the coro-
nary vessels into the sinus venosus.

The hypobranchial system of vessels is so important for the thy-
roid gland as to deserve more than passing mention. T. J. Parker
(86) has described this system in connection with his much
quoted work on the circulation in Mustelus antarcticus, but he
makes no mention of its relation to the thyroid gland, in fact, he
mentions neither the gland nor the thyroid artery; the gland
was apparently not observed.

‘The main trunk of the hypobranchial system, in the species
which I have examined is the median hypobranchial artery; it is
formed by the commissural arteries coming from the ventral ends
of the loops formed by the efferent branchial vessels which receive
blood from the gill capillaries. These loops surround each bran-
chial cleft, and within the gill they lie parallel to the afferent
branchial arteries; they are just antero-internal to the afferent
vessels. The ventral efferent vessels are very much smaller than
either the dorsal efferent or the afferent (fig. 5). Opposite the
ventral end of the second and third branchial clefts (sometimes
only the second or the third) each loop gives off a commissural
branch which passes inward and somewhat backward to the ven-
tral surface of the ventral aorta where these vessels, with consider-
able variations, unite with their fellows of the opposite side to
form a median hypobranchial artery which is frequently double
so as to form a sort of elongated arterial circle. Sometimes the
vessels fail to unite in front so that instead of a median hypobran-
chial there is a right and left hypobranchial artery, one on either
side of the ventral aorta (fig. 5). Frequently the vessels so unite
as to form an annular anastomosis which encircles the aorta,
but portions of the ring may be absent. Some of these variations
are indicated in figs. 5 and 6. The varied arrangements of these
vessels are all indications of a more or less complete fusion of the
commissural vessels to form a median hypobranchial artery. This
vessel terminates posteriorly in a small sinus-like dilatation, single
or double as the case may be. From this sinus the coronary vessels
arise either as a median vessel which promptly divides, or as two
or three independent vessels. From this same sinus a small paired

THE ANATOMY OF THE THYROID GLAND 7

=

inn
=
.<

A thyrimp

ee | -- - pharyn

gaslnic

Fig. 5. Diagram of the hypobranchialarterial circulation in Mustelus canis;
the insertions of the ventral divisions of the coraco-branchialis muscle are also
shown. Ventral view. Anastomosis with the subclavian artery was wanting
in this specimen.

178 JEREMIAH S. FERGUSON

artery passes backward on either side of the median line beneath
the dorsal portion of the pericardium at the lateral margin of the
cartilaginous floor of the pharynx formed by the basi-branchial
cartilage; after anastomosing with its fellow of the opposite side
beneath the apex of the cardiac ventricle it distributes its ter-
minal branches to the wall of the esophagus and stomach near the
cardia (figs. 5 and 6).

From the loop at the ventral end of the fourth branchial arch
a very small anastomotic branch (less frequently arising as in fig.
6, hypobr’) passes backward along the lateral wall of the pericar-
dium and penetrating between the precaval sinus and the coracoid
arch anastomoses with the subclavian artery just prior to its
division into the brachial (axillary) and the lateral (or hypo-
gastric, a large artery lying parallel to the lateral vein. This anas-
tomotic branch is undoubtedly that which T. J. Parker (’86)
describes as the hypobranchial, which, according to his descrip-
tion receives blood from the subclavian and supplies the coro-
nary arteries and whole hypobranchial system. Such is not the
case, however, in any of the species I have studied and Hyrtl
(72) in his careful study of various species of the Selachii did
not so find it, nor did Parker and Davis (99). The hypobran-
chial is a very small artery, so small that its connection with the
median hypobranchial is scarcely traceable, and insome individuals
is entirely wanting (fig. 5), there being in these cases a small
branch from the subclavian and a similar vessel from the median
hypobranchial which follow the usual course but never unite, the
subclavian branch distributing its blood to the muscles while the
anterior division supplies the lateral pericardial wall and the adja-
cent muscles in front of the coracoid arch. Certainly where the
vessel is wanting the flow of blood can not be in the direction
indicated by T. J. Parker. Parker and Davis (’99), as already
quoted, found the hypobranchial artery Be a though they
did not record its absence.

If the median hypobranchial artery of Must be injected the
major portion of the fluid passes into the coronary arteries and
thence through the coronary veins to the sinus venosus and auricle,
while at the same time very little passes through the hypobran-

THE ANATOMY OF THE THRYOID GLAND 179

chial artery; the fluid pours into the sinus venosus and auricle
very freely before it has even reached the subclavian by way of
the hypobranchial artery. This is the case whether the fish has
been previously bled or not. This experiment would certainly show

b
c0- ns ‘f.
ee core pies

epibrl | Say

-- gastric ---'

coracoid.--g

A lat---4 a =

Fig. 6. Diagram of the hypobranchial arterial circulation in Squalus acanthias.
Lateral view.

that the hypobranchial is of too small a caliber to supply the blood
necessary to fill the coronary arteries, and if it can not supply this
much it certainly is still less competent to supply blood for the
whole hypobranchial system, which T. J. Parker’s description

180 JEREMIAH 8S. FERGUSON

would seem to indicate was the case. The whole system may be
readily injected from any one of the gill-loops with which it is
connected. The true direction of flow is therefore from the effer-
ent gill-loops through the commissural arteries to the median
hypobranchial and from it to the muscular, pericardial, gastric,
esophageal, and coronary branches, with only a relatively insig-
nificant and inconstant anastomotic supply from the subclavian
artery.

Anastomoses in both the arterial and venous systems, forming
“‘cireles’’ about the body wall and the viscera are of very frequent
occurrence in this class of fishes as was pointed out for the venous
system by T. J. Parker in 1880 (vide supra). The subclavian
artery forms such a circle beneath the coracoid archand several
similar ‘‘circles” are formed by anastomosis between the two sides
in the hypobranchial system as well as in other parts of the body
with which we are not now specially concerned. The arrangement
in the arterial system is therefore very similar to that which Parker
found in the venous.

At the ventral extremity of each efferent gill-loop, at the point
where the hypobranchial commissural arteries arise, is a small
sinus-like dilatation (figs. 5 and 6, s. a. v.) which obviously serves
as a reservoir where the blood coming from the two sides of the
loop, which are in adjacent branchial arches, will intermingle, and
from this sinus blood is distributed through the commissural
arteries (‘“‘lateral hypobranchial”’ of Parker and Davis) anteriorly,
posteriorly, or to the median hypobranchial in such proportion
as the caliber of the several vessels and the course of the circula-
tion dictate. The arterial sinus at the ventral end of the first
gill-loop (first ventral sinus) is usually a trifle larger than the
others. The thyroid artery arises from the anterior end of this
sinus or from the adjacent portion of its anterior limb in the hyoi-
dean hemibranch. It arises either as a separate and independent
vessel or as a conjoined’ trunk with the mandibularartery; more
frequently, in the specimens I have dissected, it was independent.
The artery passes directly forward and inward to the extreme
lateral border or angle of the thyroid gland. It continues its
path along the surface of the thyroid gland (figs. 3, 4 and 19) near

THE ANATOMY OF THE THYROID GLAND 18t.

its anterior border, distributing its main branches to the substance
of the gland and small collateral branches to the floor of the
pharynx in front of the hyoid arch and to the anterior third of the
coraco-hyoideus and coraco-mandibularis muscles. Asmall median
unpaired vessel arising from the left thyroid artery (less frequéntly
from the right), penetrates the thyroid gland, divides, and enters
the coraco-hyoideus to supply the antero-median portion of this
muscle. This is very probably homologous with the anterior
portion of the arteria thyroidea impar, derived from the median
hypobranchial as described by Hyrtl (’72).

The left thyroid artery is usually larger, longer, and more
extensive as to its area of distribution than the right. Lombard
(09) dissected a number of specimens of Mustelus and Raia and
found that the left thyroid artery more frequently entered the
dorsal surface, and the right the ventral surface, of the thyroid
gland. I have found asomewhat similar condition, though I very
frequently find both vessels coursing upon the ventral surface
and sending their branches dorsally into the substance of the gland.
Occasionally the right thyroid artery enters the dorsal surface of
the gland and the left the ventral (fig. 3and 19). The position and
distribution of the thyroid arteries is, however, subject to consider-
able variation and, as I have already pointed out, the right may
even supply a greater portion of the gland than the left thyroid
artery (figs. 3 and 4).

The thyroid artery as described very probably in part corre-
sponds to the vessel which was recognized by T. J. Parker (’86) as
the coraco-mandibular artery. This latter is an obviously inaccu-
rate designation, for the coraco-mandibular branches are insignifi-
cant as compared with the other ramifications of the artery.
Hyrtl (’72) recognized and more accurately described the thyroid
artery as arising from the ‘‘ veins of the first gill-arch”’ in conjune-
tion with the submental artery; his description appears to be
accurate with the exception that the two arteries in my dissec-
tions appear more frequently to arise independently.

The observations which I have recorded concerning the origin

THE AMERICAN JOURNAL OF ANATOMY. VOL. 11, No. 2

182 JEREMIAH S. FERGUSON

Gaacrenaran
Ll ay Wo)
Meas el i
Le eid
KUASERSS

tilt i }

We ni He

Nig. 7. The position and relations of the thyroid gland in Raia.

Fig. 8. Injected lymphatics in the tunica adventitia of the right commissural
artery at its junction with the median hypobranchial. The specimen is from
Carcharias, the fish having been injected with ink at a point just ventral to the
right hyoidean hemibranch. The injection followed the lymphatics and was

traced as far as the median hypobranchial artery in one direction and into the
thyroid gland in the other.

THE ANATOMY OF THE THYROID GLAND 183

and course of the thyroid artery and the hypobranchial system
were carefully worked out with specimens of Mustelus and Squa-
lus and verified in all their important particulars in Carcharias
and Raia. ‘

VEINS AND LYMPHATICS OF THE THYROID REGION

The numerous small veins of the thyroid gland discharge into
the ‘‘thyroid sinus,’”’ which connects together the hyoid sinuses of
the two sides. The conformation of the hyoid sinuses and their
tributaries and connections have been well described by T. J.
Parker (’86). In addition to the transverse anastomosis formed
by the thyroid sinus, the hyoid sinus receives a submental vein
from the region of the mandible and numerous small muscular
branches from the neighboring muscles. This sinus and its con-
necting vessels can be most readily observed in Raia. The sub-
mental vein is seen to begin as a double transverse anastomosis;
the larger, anterior, tributary lies close behind the cartilage of the
inferior mandible; the smaller, posterior vessel arches across the
floor of the mouth just in front of the hyoid arch and the anterior
border of the thyroid gland. At the angle of the jaw these vessels
unite in a small sinus which also receives a transverse anastomo-
sis from in front of the maxilla, so that the mouth is thus encircled
by an annular venous sinus. The thyroid sinus similarly forms a
double transverse anastomosis, rather more deeply placed, behind
the hyoid arch at the posterior border of the gland. These vessels
convey the blood from the ventral cervical region to the hyoid
sinus.

The thyroid veins open into the thyroid sinus as several small
branches, the largest of which are a median vein, leaving the organ
near the middle of its dorsal surface, and two anterior veins which
leave the same surface near the anterior margin of the organ,
but a little to either side of the median line. Other smaller veins
leave the lateral margins of the organ passing either to the thyroid
or the hyoid sinus. The thyroid veins must contain valves, for
although the vessels can be readily traced with the dissecting
microscope and even with the naked eye, it is with difficulty that

184 JEREMIAH S. FERGUSON

fluid injected into the thyroid or hyoid sinus can be forced back
into the venous channels of the thyroid gland; an extreme pres-
sure will accomplish this result to a limited extent only. I have
been able to find some traces of valves in microscopical sections.

The hyoid sinus passes around the base of the hyoidean hemi-
branch to connect with the jugular vein, through which a portion
of its blood is transmitted to the precaval sinus beneath the cora-
coid arch, and thence to the sinus venosus and auricle. The flow
through the hyoid sinus in this direction is quite intermittent,
_ and, as already indicated, it is chiefly dependent upon the muscu-
lar force of the pharynx as it alternately relaxes and contracts to
force water through the gill-openings.

Blood is also transmitted from the hyoid sinus to the heart by
the more ventral and direct path through the inferior jugular
(anterior cardinal) vein. This vessel maintains a more constant
flow, receiving blood from the ventral cervical region and the
branchial arches, along the ventral ends of which it courses to
terminate in the precaval sinus:

In Raia the thyroid sinus is thin, and its investment of connec-
tive tissue containing the lymphatic plexus is less pronounced than
in the other species studied, so that, except when the sinus is
fully distended with blood, the gland in Raia isnot much obscured.
In Mustelus the sinus is larger and the fascia about it is more
voluminous so that the gland is usually more or less obscured,
though there is much individual variation: the same is true of
Squalus. In Carcharias the vascular walls in the sinus are so
thick, and the connective tissue about it so abundant that in most
of the animals examined the outline of the thyroid gland con-
tained within this mass could only be discerned on holding up
the stretched membranous mass between the eye and the bright
sun so that the intense transmitted light showed the yellowish
orange gland contained within the connective tissue mass.

The thyroid lymphatic plexus forms an extensive group ot
vascular channels surrounding the gland and the vessels of the
blood sinus. It is contained in a fold of the deep cervical fascia
which stretches across from side to side between the ventral
ends of the first branchial clefts: it is broad in the mid-portion,

THE ANATOMY OF THE THYROID GLAND 185

but tapers from the postero-lateral angle of the thyroid gland
outward to the tissue surrounding the hyoid sinus. The vessels
form perivascular lymphatics about the venous sinuses. Ink or
a colored fluid injected into the connective tissue about the lyoid
and thyroid sinuses readily fills the anastomosing vessels forming a
sheetlike mass of peculiar form (fig.2, thyr.sn.). Ink thus injected
can also be traced into the perivascular lymphatics of the hypo-
branchial arterial vessels (fig. 8) as far backward as the walls of the
coronary arteries; it can likewise be found in small perivascular
lymphatics in the walls of the thyroid arteries and to some extent
in the broad venous spaces between the vesicles of the thyroid gland,
indicating that the lymphatic vessels to some extent may open into
the veins of the thyroid. The vessels of the lymphatic plexus in
the cervical fascia are apparently connected with the blood-vessels
of the thyroid sinus, for excessive pharyngeal contraction in the
living fish forces blood into areas which otherwise appear to be
occupied only by lymphatic vessels. The blood-vessels may with-
out doubt be classed as ‘‘venae lymphaticae”’ and the lym-
phatics as vasa lymphatica”’ after the terminology of Favaro
(06), who says that the same vessel may in fishes carry either
blood or lymph at the same or different times so that these vessels
may in this sense be either vasa or venae lymphaticae. Fluid
injected into the lymphatics spreads so rapidly over so great an
area that it seems almost impossible to trace a connection with
the blood sinus by means of injections; the fluid enters the blood-
vessels so readily that one is unable to exclude the possibility of
an intra-venous injection.

The statement by Baber (’81) that he was able to demonstrate
no lymphatics in the thyroid gland of Elasmobranchs led me to
pay special attention to the study of these vessels by injection
methods. As I have already pointed out, Baber states that ‘‘in
both the skate and the Conger-eel an extensive system of vessels
lined with epithelium becomes injected by the method of punc-
ture.” He then injected the blood vessels of a Conger-eel with
Berlin blue through the ‘‘efferent branchial vein” and“‘dorsal
aorta and thereupon states that ‘‘in the Conger-eel at least, there
is no evidence of any system of lymphatic vessels,’’ emphasizing

186 JEREMIAH S. FERGUSON

his statement by the use of italics. I can confirm that portion of
Baber’s statement which says that an extensive system of vessels
within the thyroid gland can be readily injected by the method of
puncture, but I would maintain that neither that procedure, nor
the injection of the dorsal aorta or efferent branchial vessels
with Berlin blue, would demonstrate the absence of lymphatics;
that they may still be present, I have demonstrated both in micro-
scopical sections and by injection (figs. 9, 10 and 11).

Fig. 9. Lymphaties, ‘“‘vasa lymphatica,’’ and veins, ‘“‘venae lymphaticae,”’
of the thyroid gland. The ‘vasa lymphatica’’ have been injected with ink and
the thyroid gland cleared and mounted in toto; the lumen of the follicle and the
follicular epithelium are only indistinctly seen. At X in the specimen the two
sets of vessels anastomose.

Injection by puncture does not always fill the extensive system
of vessels observed by Baber. If one takes care to use only a very
gentle pressure, this system, which completely surrounds each
vesicle, is only filled near the point of injection, while at the mar-
gins of the injected area the fluid spreads through more minute
vessels which lie in closer contact with the vesicular epithelium
(fig. 9,1). I believe these last are true vasa lymphatica in the sense

THE ANATOMY OF THE THYROID GLAND 187

of Favaro and I find the contents of the vesicles. apparently
secreted into them as in the mammalian thyroid (fig. 10). The
larger vascular channels then are venae lymphaticae, readily in-
jected by puncture if the pressure is excessive, the veins being
easily entered because of their large caliber and extremely thin
walls; they transmit only lymph when the intravenous blood-
pressure is low within the gland, but fill with blood when from
any cause the pressure is raised. I have invariably found
some blood cells in the venae lymphaticae; I have never found
them filled with blood in all the three score animals I have
examined except in one case in which as a result of injury the thy-
roid gland was greatly congested. In this case they were filled to
distension. In microscopical sections I have been able to trace
the connection of the vasa with the venae lymphaticae (fig. 10).
I have been unable to demonstrate positively the presence of any
valves at the orifices of these vessels, but the extreme obliquity
of the anastomosis considered in conjunction with the very thin
vascular walls might well serve a valvular function when the blood
pressure is low, though with increased pressure and venous dis-
tention some blood would be forced back into the vasa lymphaticae
and even into the vesicles. The frequent occurrence of red blood
corpuscles within the vesicles of all animals is well known and in
the Elasmobranchs it is thus accounted for. The intimate rela-
tion between the venous and lymphatic systems pointed out by
Sabin (’09) would possibly suggest that an homologous vascular
relation may account for the presence of red blood corpuscles
within the vesicles of the mammalian thyroid gland.

THE HISTOLOGY OF THE ELASMOBRANCH THYROID GLAND

The thyroid gland in Elasmobranchs consists of a mass of ve-
sicular follicles (figs. 10, 11 and 13 to 18) which very closely re-
semble those of the mammalian gland. The vesicles are lined by
epithelium of a low columnar type, contain more or less colloid
material, and are loosely bound together by a connective tissue
framework which is very richly supplied with blood-vessels.

The shape of the gland in Mustelus canis (fig 1,C) is sufficiently

Fig. 10. Section of the thyroid gland of Mustelus canis showing the anasto-
‘mosis at the point X of the vasa and venae lymphaticae. '

Vig. 11. Typical section of the thyroid gland of Mustelus canis; the intimate
relation of the epithelium of the thyroid follicles to the lymphatics and blood ves-
vels is accurately shown.

THE ANATOMY OF THE THYROID GLAND 189

peculiar to deserve passing mention. It may be described as con-
sisting of two triangles whose bases are fused in the median line,
the apices directed outward, the anterior borders convex and con-
forming to the anterior margin of the basi-hyal cartilage, the
posterior borders concave and free, except for their attachment to
the deep cervical fascia. In the median line the conjoined bases
are prolonged backward to form a short median projection;
anteriorly a shallow notch separates the two lateral triangular
hatves. The gland is approximately bilaterally symmetrical
(figs. 1,3 and 4).

The thin, almost membranous character of the gland in Muste-
lus canis offers an excellent opportunity for the recognition of a
lobar or lobular structure if such exists, for the whole gland
is frequently no more than four or five follicles in thickness and
may be stained, cleared and mounted zn toto, giving very excel-
lent microscopical pictures of the entire organ. I have not been
able to find any indication of definite lobes or lobules. Portions
of the thyroid substance are here and there wanting, as observed
by Lombard (09), and these deficient areas occur more frequently
in the posterior than in the anterior half of the gland. In one of the
thirty-two fishes of this species the deficiencies were so great that
the gland was only represented by a few specks which were posi-
tively identified as portions of the thyroid only after microscopical
examination. A similar case was found in Squalus, and one gland
from Carcharias consisted of three small pieces.

Occasionally the posterior border presents a notched deficiency
in or near the median line. There may be one, two or three such
notches, either symmetrically or asymmetrically disposed. Defici-
encies of the thyroid tissue also occur within the gland and may,
or may not, be connected with the notches in the posterior border.
These deficiencies are all of inconstant occurrence, irregular loca-
tion, and could scarcely be taken to indicate any suggestion
of definite lobes. They seem rather to be due to the extreme
thinness of the gland and in many of the thicker specimens they
are in no way indicated. When present they are occupied by con-
nective tissue continuous with the glandular capsule. Frequently
they transmit the larger thyroid vessels.

190 JEREMIAH 8S. FERGUSON

Bits of thyroid tissue of inconstant form or location are occa-
sionally separated from the body of the gland by narrow partitions
of connective tissue; they are most frequently found near the
border of the gland or adjoining an area in which the thyroid sub-
stance is: deficient. Since they possess no constant relation
to the vascular supply, the detached masses can not correspond in
any sense to true anatomical lobules. The arteries branch irregu-
larly, for the most part after a somewhat dicotymous fashion
(fig. 12), the arterial twigs passing off at acute angles. Partially
injected specimens in which the injection fluid has passed through
the arteries but has not penetrated in quantity into the veins

Fig. 12. Terminal divisions of the thyroid artery. The area occupied by
injected capillaries, ‘‘venae lymphaticae,’’ directly connected with each terminal
arteriole is roughly indicated by the dotted lines.

show areas of injected capillaries surrounding the terminal arteri-
oles (fig. 12), but the extent of these injected areas and their
relation to the artery seems to be dependent rather on the pressure
of the injection than on any constant or characteristic relation to
the vascular system. I can not recognize any probable vascular
or anatomical unit which might in any sense serve as an ana-
tomical lobule or structural unit, as described for various other
glands by Born, Mall and others.

In Raia the occurrence of partially detached groups of thyroid
follicles is more frequent than in the other species, but the number
of such groups present in a gland varies from two or three to a
score or more. The groups are outlined by connective tissue in

THE ANATOMY OF THE THYROID GLAND 191

which broad venous spaces to a certain extent encircle the quasi-
lobule. The veins thus lie at the periphery while the artery on
reaching the group promptly breaks up into a plexus of broad
capillary spaces—venae lymphaticae—which surround the folli-
cles within the quasi-lobule. The number of follicles in the group
varies from four or five to several score.

In Carcharias the condition is similar to that in Mustelus, there
being no indication of lobular groups except about the occasional
irregular deficiencies in the thyroid mass. Except for the anatom-
ical disintegration of the gland in one fish there was similarly no
indication of lobulation in Squalus, but as none of my specimens
from this species were prepared as total mounts I can not speak
with the same certainty as in the other species.

The form of the thyroid follicles is subject to considerable
variation, but, in general, they may be said to be of ovoid shape,
and, as pointed out for the mammalian thyroid by Streiff (’97),
they present frequent diverticula. The Elasmobranch thyroid
differs from those described by Streiff in that they show very little
tendency to branch and no indication of a tubular character when
the whole follicles are examined in total mounts of the gland (fig.
13). In cut sections diverticula are of frequent occurrence and are
apparently the result of pronounced infoldings of the follicular
wall rather than of any protuberance, or of any tendency of the
follicle to branch. Fig. 13 shows characteristic follicles from all
four species; the figures are of whole follicles and differ from the
cut sections in that only the largest infoldings of their wall are
visible. As already indicated, diverticula are more apparent in
sections than in the preparations (total mounts) from which the
drawings have been made. The particular follicles drawn from
Carcharias present rather greater infoldings than those from the
other species. I have not, however, observed that this is charac-
teristic of Carcharias. In the figure the magnification is the same
for the several Selachian species but less by one half for Raia; the
follicles of Raia are, therefore, relatively about twice as large as
shown.

The size of the follicles is subject to considerable variation as
regards the individual follicles, the different thyroids, and the

192 JEREMIAH S. FERGUSON

Fig. 13. Outline of the follicles of thyroid glands as seen in total mounts. A,
from Mustelus canis, X 152. B, from Squalus acanthias, X 152. C, from Car-
charias litoralis, X 152. D, from Raia erinacea, X 80.

various species. I have tabulated the results of some of the meas-
urements.

| MAXIMUM MINIMUM AVERAGE ‘|| AVERAGE RATIO ehcp
SPECIES DIAMETER OF DIAMETER OF DIAMETER OF LENGTH OF | FOLLICLE TO THYROID
FOLLICLE FOLLICLE | FOLLICLE FISH FISH GLAND
| | 7
Mustelus........ | .160mm. | .017mm.| .067mm.| 67.8cm. 101.7) | 136mm;
Squalus...... ay 079 | .013 047 oem Te no data
Carcharias....... Dts | 0238 .100 | NT a2, Tee eh USI cf
o © | ~ P |
FUAYAA Nene el ao | .053 . 167 46.2 PAL atl 6.5

THE ANATOMY OF THE THYROID GLAND 193

The very large relative size of the follicles of Raia is at once
apparent. They are approximately four times as large, relatively
to the length of the fish, as in the case of any other species. When
compared with the diameter of the gland the ratio is again in-
creased, but this difference is in part compensated for by the
increased thickness of the gland in Raia as compared with the
other species. The thyroid gland of Raia is 1.5 times as thick as
that of Carcharias, and 2 to 2.5 times the thickness of the gland
in Mustelus.

The column of ratios in the above table would indicate that in
the Selachians the size of the follicle is in approximate proportion
to the size of the fish, but that in Raia the relative size of the
follicleismany times as great; the actual number of follicles in the
thyroid gland of Raia is only a small fraction of those in the gland
of any of the other species. It is readily susceptible of mathemat-
ical proof that the combined circumference of large follicles con-
tained in a given area is less than the combined circumference of
smaller follicles in the same area; hence the gland of Raia with its
larger follicles will contain proportionately less epithelium than
the glands of the other species. I estimate that the difference
is just about sufficient to render the volume of secretory epithelium
in the gland of Raia relative to the size of the fish equal to the
volume of secretory epithelium in each of the other species.

But it is equally susceptible of mathematical proof that the
cubical contents of the combined follicles is greater in the
gland having the larger follicles; hence there is in Raia a greater
volume of intrafollicular space than in the other species. It is
scarcely susceptible of proof but entirely reasonable to suppose
that the epithelium of different individuals of the same or different
species so closely allied and the Batoidei and the Selachii is approx-
imately equally active as regards its secretory function. There is
ample evidence that the fluid secreted by the thyroid epithelium
into the cavity of the follicle finds its way through the wall of the
follicle to the neighboring vascular spaces so that the direction of
flow must, in part at least, be from the epithelium into the follicle
and thence through the follicular wall to the vessels. In view of
these facts the rate of this secretory flow in Raia, with its relatively

194 JEREMIAH S. FERGUSON

large intrafollicular space must be slower than in the other spe-
cies with their relatively small intrafollicular cavities, or, to ex-
press it differently, there is relative stagnation in intrafollicular
secretory flow in the case of the thyroid follicles of Raia. It is
well known that an albuminous secretion which is rendered rela-
tively stagnant within the epithelial cavities of the body tends to
produce colloid masses whose microchemical reactions more or
less closely resemble those of the colloid material of the thyroid
gland; this occurs, e.g., in the ducts and tubules of the resting
mammary gland and in dilated cystic tubules in the kidney. We
would therefore expect that in the thyroid follicles of Raia with
their relatively stagnant secretory flow we should find an increased
amount of colloid material. This I find to be the case, the pro-
portionate volume of colloid present in the follicles of Raia being
decidedly greater than in the other species. Similarly I find it
the rule that the larger follicles contain relatively more colloid
than the small follicles in the same gland. The volume of col-
loid contained in the thyroid follicles, therefore, can not be
regarded as an. index of the activity of the secretory epithelium;
it would rather appear as a sort of by-product whose volume
was dependent upon the rate of flow in the fluid from which it
was formed. This view harmonizes the appearance of colloid
material in the thyroid gland with the occurrence of similar mate-
rial in the other glandular portions of the body, and with those
theories of thyroid secretion which regard the colloid as a by-
product rather than as the secretion. Moreover the great varia-
tions in the amount of colloid in the thyroid follicles are then
explicable upon the basis of variations in the rate of secretory
flow which, in turn, is dependent upon the physiological factors of
blood and nerve supply as well as upon the anatomical factors.

It is also interesting to observe that the volume of secretory
epithelium in the several species examined remains in each case
approximately proportionate to the size of the thyroid gland and
to the size of the fish. The relation of the epithelium to the folli-
cular content and to the blood vessels and lymphatics seems to me
to indicate most clearly that the secretion is poured out from the
epithelial cells so as to find its way, on the one hand directly into

THE ANATOMY OF THE THYROID GLAND 195

the vessels, and on the other hand into the follicular cavity, whence
it eventually passes through the follicular wall to reach the vessel.
It is in the course of the latter flow that the colloid appears and
its volume is dependent upon the rate of flow.

The question arises as to whether or not the colloid may serve
for the storage of secreted materials, somewhat after the manner
in which the hepatic glycogen may be considered as stored car-
bohydrate to be delivered as the needs of the economy necessitates ;
if so the colloid material should show further evidences of change,
at least, under certain conditions. That the colloid does undergo
changes is evidenced by the appearance within its otherwise
homogeneous mass of such structural alterations as vacuolation,
basophile degeneration, and disintegration into granules of greater
or less size, changes which are frequently observed (figs. 14, 15 and
18). As to the physiological nature of these changes in colloid,
and their possible connection with a storage function I can offer
no conclusive proof, but it seems to me quite possible that such
a relation exists.

The thyroid follicles are lined by a simple columnar type of
epithelium (fig. 11) whose cells show considerable variation in
height. In the same gland the epithelium lining certain vesicles
measured as much as .010 mm., others only .006mm. The epi-
thelium of occasional vesicles was even lower, but was possibly
open to the criticism of mechanical distortion since the colloid
was often crowded against one side instead of lying in the middle
of the follicular lumen, even though the tissues had been prepared
with the greatest care. Being anxious to avoid any possible
distortion of the tissue, I removed nearly all of the glands
studied without allowing them to be touched by either instru-
ment or fingers, the knife or scissors being passed through the
muscle beneath, and the gland, supported on a thin layer of
muscle, dropped bodily into the killing fluid.

As a rule those follicles which were well filled with colloid
possessed low epithelium, in those with taller epithelium the reverse
was the case. In making this comparison the surface area of the
sections of colloid mass was compared with that of the containing
follicle. The average height of the epithelium of a number of fol-

196 JEREMIAH S. FERGUSON

licles in Mustelus which were well filled with colloid was .077 mm..,
while in a similar number of follicles which were either devoid of
colloid, or nearly so, the height of the epithelium averaged .087 mm.
Each epithelial cell possesses a fairly distinct cell-wall. Many
cells appear to have a well marked cuticular border which appears
to be more highly refractive than the endoplasm, but wherever the
colloid lies in contact with the surface of the cell the cuticular
border is obscured. I have also noticed that it is less pronounced
in the thinner sections so that I am inclined to regard it as an
optical diffraction line rather than a true cuticular membrane.
The conformation of the free ends of the epithelial cells tends to
confirm this opinion. These cells project slightly into the lumen of
the follicle by means of a somewhat convex free border so that the
height of a cell is greater in its axis than at its margin. Thus the
lower portions of a cell will, in the thicker sections (.010 mm. or
more), show through the taller central or axial portions and so
account, at least, for a portion of the cuticular appearance.
The exoplasmic membrane is specially distinct in the epithe-
lium of the Elasmobranch thyroid gland. Baber (’81) called atten-
tion to this fact, and described it as an intercellular network
enveloping the cells and connecting the lumen of the follicle with
the surrounding tissue spaces. If the lining epithelium of the
follicle be cut parallel to the surface the resulting sections will
show the membrane as a distinct’ mosaic within whose meshes the
cells are apparently contained. It appears to me that this mosaic,
which is distinct from the intercellular colloid observed by Lang-
endorf (see page 200), is rather to be regarded as a cell membrane
than as an intercellular substance, for there are many portions
where in thin sections a narrow intercellular space is distinctly
apparent and is bounded on either side by the exoplasmic mem-
brane of adjacent cells. Occasionally the cells are separated by
wider intervals through which the follicular lumen is placed in
direct communication with the surrounding tissue spaces.
There appears to be no distinct basement membrane upon
which the follicular epithelium may rest. At intervals the cells
are invested with a very small amount of loose connective tissue
(fig. 11), but in large part the epithelium rests directly upon the

THE ANATOMY OF THE THYROID GLAND 197

walls of the venous channels and lymphatic vessels (fig. 14).
Thus the relation of the epithelium to the vascular lumen is a
very intimate one. ;

The cytoplasm of the ‘“‘chief”’ cells is relatively clear, but con-
tains a coarsely granular eosinophile reticulum. Some cells
appear much more granular than others. In such cells as are filled
with colloid, ‘‘colloid cells,” the granular reticulum is entirely
obscured (fig. 14, A).

The nuclei of the chief cells are spheroidal, vesicular, and are
placed near the base of the cell. From the apices of many of these
cells threads of secretion extend to the central colloid mass. The
apices of many of the chief cells appear ragged, frayed, and often
shrunken, so that the height of the cell is decreased. Such cells
present an appearance suggestive of an advanced stage of secre-
tion. Other cells contain granules at the distal ends which are
arranged in vertical rows, giving this portion of the cell a some-
what rodded appearance; such cells are usually well filled with
granules. Occasionally a similarly ragged and rodded appearance
is seen at the base of the cell and it suggests that secretion may
also be discharged at that point. Such a possibility is rendered
more probable by the absence of basement membrane and the
intimate relation to the lymphatics and blood vessels, these cells
often resting directly upon the vascular endothelium. Laterally
the epithelial cells frequently are separated from one another,
leaving considerable spaces or channels through which secretion
may find its way from the follicular lumen to the neighboring
vessels; such channels are often occupied in part by colloid and in
a few cases I have traced the colloid in a continuous line from the
intrafollicular mass to the interior of the vasa and venae lympha-
ticae (fig. 10).

The above observations suggest that secretion may either be
discharged from the chief cells into the lumen of the follicle
and thence find its way through the follicular wall to the blood
and lymphatic vessels, or that it may be discharged from the cells
directly into the vessels; this is in harmony with the conditions
indicated in the thyroid gland of mammals.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 11, No. 2.

198 JEREMIAH 8S. FERGUSON

Fig. 14. A, section of the thyroid gland of Mustelus showing a follicle com-
At y a carmine granule, derived from the
B, section of the thyroid gland

of Mustelus showing follicles lined entirely by “‘chief cells.”’

pletely surrounded by ‘‘colloid cells.’’
injection mass, lies in the vena lymphatica.

THE ANATOMY OF THE THYROID GLAND 199

The colloid cells described by Langendorf (’89) are remarkably
distinct in most of the sections of Elasmobranch thyroids and con-
stitute one of the most characteristic features of the thyroid gland
of these fishes. The colloid cells are distinctly acidophile and are
easily recognized in specimens stained with hematoxylin and
eosin if the eosin is used in dilute solution and allowed to act for
one-half hour or more. They present a glistening, highly refractive
colloid appearance, which is in marked contrast to the granular
chief cells. The colloid cells occasionally occur singly, but aremore
frequently disposed in groups along one side of the follicle. One
such group (fig. 14, A), more extensive than the others, was seen
to include fully three-fourths of all the epithelium in its follicle.
The groups are often in contact with the central colloid mass, and
the colloid within the cell may then appear continuous with that
within the follicle. Occasionally a group of such epithelium
appears to have been completely engulfed by the colloid mass, the
epithelial nuclei then appearing well within the colloid. Such
appearances might suggest mechanical distortion, but as the sur-
rounding follicles show no evidences of injury, and, as already
stated, the tissues were very carefully prepared, I am more in-
clined to agree with Bozzi (’95) that these appearances are the
result of vital phenomena.

The nuclei of the colloid cells are small and deeply stained, so
deeply, in fact, that in the usual preparations they frequently
show neither nuclear wall nor karyosomes. Unlike the nuclei
of the chief cells they are usually situated near the inner extremity
of the cell rather than at its base. The greater the cell is distended
with colloid the farther its nucleus is pushed toward the cell’s
apex; in the most distended cells there was frequently some dis-
tortion and even fragmentation of the nucleus. A further continu-
ation of this process would account for at least a portion of the
extruded and disintegrating nuclei found within the intrafollicular
colloid masses (figs. 11 and 18).

The intrafollicular colloid closely resembles that of the mamma-
lian thyroid gland. It is strongly acidophile and is usually homo-
geneous or very finely granular in appearance. Frequently a
minor portion of the mass, e.g., one side, is finely granular while

200 JEREMIAH S. FERGUSON

the major portion is clearly homogeneous. The well-known fila-
ments pass at frequent intervals from the colloid mass to the
epithelial surface. Some of these filaments can be traced to the
free surface of the epithelial cell while others quite clearly enter
the intercellular spaces, where, in tangential sections of the fol-
licle, they form intercellular masses simulating the net-work de-
scribed by Baber (’81) and interpreted by Langendorf (’89) as
the ramification of colloid cells.

Occasionally the colloid mass appears to have been disinte-
- grated into small spherules .007 to .008 mm. in diameter (fig. 15).
The size of these spheres is suggestive of the red blood cells of
mammals, but the red cells of Elasmobranchs are ovoid and larger.
Of the spherules some are distinctly acidophile but many are
slightly basophile, none, or very few, are strongly basophile. All
the spherules are homogeneous, and I have observed in the more
basophile no tendency to chromatolysis such as one might expect
to find if the spherules of this type were thought to represent de-
generating nuclei of the red cells, nor have I been able to trace
stages of transition from the nucleus to the basophile spherule.
Since all the blood cells-of the species studied are nucleated one
could not well infer that the acidophile spherules could represent
any stage in the disintegration of red blood cells, for none of these
spherules contain even traces of chromatin. On the other hand,
both red and white blood cells can occasionally be found within the
colloid quite independently of the spherules I have described;
in this particular the Elasmobranch thyroid is in accord with the
well known structure in other vertebrate orders. The appear-
ance, location, disposition and reactions of the spherules indicate
their origin from the solid colloid masses, from which they would
appear to be formed by disintegration with progressively increas-
ing basic reaction. That the reverse process occurs, viz., that the
spherules may represent intermediate stages in the formation of the
colloid masses, is contraindicated by the fact that only very few
follicles contain spherules, nor does there appear to be any indica-
tion of a tendency of the spherule to fuse. On the other hand, a
tendency to further disintegration is quite apparent, and the
possibility is suggested that the colloid in this way may be trans-

THE ANATOMY OF THE THYROID GLAND> 201

Fig. 15. Section of the thyroid gland of Raia showing the colloid within a
follicle disintegrated into spheroidal masses of varying size and depth of stain.

Fig. 16. Section through the ventral margin of the thyroid gland of Mustelus
with sections of the very broad venae lymphaticae of the thyroid sinus simulating
an endothelial capsule about the gland.

202 JEREMIAH S. FERGUSON

formed to such a state that it may be secreted through the wall
of the follicle, in which case the intrafollicular colloid would pre-
sumably assume the nature of stored secretion which is first poured
out from the cells as a fluid, is then condensed through retention
within the follicle, to form colloid, and is later disintegrated, pass-
ing out of the follicle with the secretory flow. The chemical analy-
ses of the thyroid gland, showing the common relation of iodin
to the colloid and to the active principle of thyroid secretion
would harmonize with such an hypothesis.

Vacuolation of the colloid mass (fig. 14) is of frequent occur-
rence; it may result either from the inclusion within the intra-
follicular colloid of portions of the peripheral cup-like impressions
which Langendorf has surmised result from the secretion pouring
out from the surface of the epithelial cells, or it may be further
evidence of disintegration of the colloid with formation within
its substance of fluid droplets, rather than of solid spherules.
The vacuoles are filled with a clear fluid and occasionally contain
basophile granules. A colloid mass may contain many small
vacuoles mostly at or near the periphery of the mass and contain-
ing few, if any, basophile granules, or it may contain one or more
vacuoles of relatively large size which occupy the interior of the
mass and may be more or less completely filled with the granules.
These granules stain deeply with hematoxylin and similar dyes,
and they are either amorphous or somewhat crystalline in form.
The origin of the chromatic material within the vacuoles may be
from chromatolysis of the nuclei of either the disintegrating folli-
cular epithelium or of blood cells included within the colloid.
Evidences of disintegration of epithelium and extrusion of the
nuclei as well as of the penetration of the nuclei together with
blood cells (fig. 20) into the colloid mass are frequently seen. But
the disintegration of such cells and nuclei can scarcely account
for the much more numerous vacuoles in which no basophile
chromatic substance is found.

The follicles of the thyroid are supported within the meshes of
a connective tissue stroma in which the blood-vessels lie. The
volume of connective tissue is never great, much less than in the
mammalian gland. There is more connective tissue in the gland

THE ANATOMY OF THE THYROID GLAND 203

of Raia than in the other species examined; in Mustelus and Squa-
lus there is so little that one wonders at the relative compactness
of the organ. In these Selachii the epithelium rests directly upon
the walls of the blood-vessels, and they in turn consist of little
else than endothelium and form broad sinuses rather than capil-
laries or venules. ,

The gland is inclosed by a very thin connective tissue capsule
and its tissue is thus always sharply defined from the surrounding
structures. In Mustelus and Squalus, and to some extent in the
other species, the broad vascular channels of the thyroid sinus
are in direct contact with the capsule, so that in sections the ven-
tral surface of the gland often appears clothed with an endothelial
coat derived from these vessels (figs. 16 and 17); a similar disposi-
tion of the vascular endothelium of collapsed blood-vessels is also
occasionally seen on the margins and dorsal surface of the gland.

The blood vessels have been in each case carefully studied by
dissection, injection, sections, and transparent total mounts of
the gland. Both blood vessels and lymphatics were demonstrated
beyond doubt, though lymphatics have not hitherto been observed
in these fishes and their existence was denied by Baber (’81).
Fig. 9 shows the lymphatics filled with injection mass, lying
between the blood channels and the follicular epithelium; they
appear as perivascular lymphatics in the wall of the venae lym-
phaticae. Similar vessels, perivascular lymphatics, are found in
the walls of the arteries and veins of the thyroid, the thyroid sinus,
and the arteries of the hypo-branchial system (fig. 8).

The course of the larger blood-vessels was readily followed
in injected specimens in which the whole gland was examined
under the microscope. The arteries course upon the surface of
the gland, the major portion of them being always on the ventral
surface. Fig. 19 shows the distribution of the arteries in the thy-
roid of Mustelus, and figs. 3 and 4 indicate the relative area of the
gland supplied by the arteries of the right and left side, the left
thyroid artery, as in figs. 3 and 19 usually supplying the greater
part of the organ, though occasionally the major part, as in fig. 4, is
supplied from the right side. Twigs from the superficial branches
here and there penetrate the gland, break into arterioles, and

204

JEREMIAH 8. FERGUSON

Fig. 17. Section through the ventral margin of the thyroid gland of Squalus
showing peripheral venae and vasa lymphatica.

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Fig. 18. Section of the thyroid gland of Carcharias showing the disintegrating
nuclei of leucocytes or red blood cells within the intrafollicular colloid.

THE ANATOMY OF THE THYROID GLAND 205

promptly empty into groups of broad interfollicular, blood capil-
laries, the venae lymphaticae (figs. 9, 11, 12), which envelop the
follicles on all sides. From the venae lymphaticae the veins pass
out of the gland at its posterior and lateral borders and dorsal
surface to enter the thyroid sinus; a few veins from the lateral bor-
der of the gland pass directly to the hyoid sinus.

The course of the lymphatics was much less easily determined
than that of the blood vessels. “Stick injections,” as ordinarily
made, spread so rapidly through the loose connective tissue of
the gland and so easily entered and filled the venae lymphaticae
that they entirely obscured the smaller vasa lymphatica. The
venae lymphaticae thus injected form a dense almost opaque mass,
showing that the thyroid may well occupy the place in these fishes
assigned to it by Tscheuwsky (’03) as the most vascular of mam-
malian glands. After several futile attempts to inject the lym-
phatics in the ordinary way the method was so altered as to inject
only minute areas under a very low pressure. In this way it
was found that at the margins of the injected area the fluid which
had entered the vessels traveled farther than that in the connec-
tive tissue spaces, and in many cases the vasa lymphatica were
filled beyond the limits of the injected venae lymphaticae, so that
in the outermost zone of the injected area the true lymphatics
could be readily studied, the venae lymphaticae in this zone being
either empty or only partially filled.

The vasa lymphatica are, for the most part, perivascular chan-
nels (fig. 9), but they are also in direct contact with the epithelial
walls of the follicles (fig. 10). The vasa lymphatica could not
be followed for any great distance through the injected zone, for,
on the one hand, they entered the area of opaque injection mass,
and in the other direction they ended abruptly, often with a
small knob-like dilatation. By means of serial sections I was able
to determine in uninjected specimens that the vasa lymphatica
opened at the points of terminal dilatation directly into the venae
lymphaticae (fig. 10, X). Having demonstrated the connection
between the two sets of vessels in uninjected specimens, show-
ing the true relation of vasa and venae lymphaticae, many
points in the injected specimens could be readily found at
which it seemed quite certain that the injection mass was

206 JEREMIAH S. FERGUSON

art. thyrsinestra
19

Fig. 19. Drawn from a total mount of the thyroid gland of Mustelus canis,
showing the course and distribution of the thyroid arteries and the origin of some
of the veins of exit. The vessels on or near the ventral surface are indicated by
the solid black lines, those on or near the dorsal surface of the gland by dotted
lines.

Fig. 20. A section from the thyroid gland of Carcharias, showing invasion of
epithelium and colloid by leucocytes.

THE ANATOMY OF THE THYROID GLAND 207

passing from the vasa lymphatica directly into the venae. The
relatively intimate relation between the veins and lymphatics
in fishes is well known; Wiedersheim (’07) and Favaro (’06)
have recently emphasized the fact so far as the tail vessels of
fishes were concerned. ‘This intimate relationship seems to be
quite as obvious in the thyroid vessels and in those of the region
occupied by the thyroid sinus (vide supra).

That the vasa lymphatica are true lymphatics and not blood-
vessels is shown by the fact that in many cases they are of alto-
gether too small caliber to transmit the large red blood-cells of the
Elasmobranch fishes. Moreover, the quasi valvular nature of
their anastomosis with the veins, as already described, renders
highly improbable the regurgitation of blood-cells into the vasa
lymphatica even in the larger vessels.

In mammalian thyroid glands, especially in dogs, one now and
then observes instances where the colloid has accumulated beyond
the bounds of the follicles, giving to sections of the organ the
appearance of a tissue completely infiltrated by the waxy colloid
substance. No such appearance was found in the Elasmobranch
thyroids which were studied, unless the follicles lined chiefly
by colloid cells could be so interpreted.

SUMMARY

1. The Elasmobranch thyroid gland closely simulates the
human, both in the form and structure of its follicles and the dis-
tribution of its blood-vessels.

2. The gland rests upon the basi-hyal cartilage whose anterior
margin forms an excellent guide to its location.

3. The pyramidal lobe of mammals is often represented in Elas-
mobranchs by a process passing forward and reaching the floor
of the pharynx through a notch in the anterior margin of the basi-
hyal cartilage; this notch is sometimes converted into a foramen.

4. Baber’s opinion that lymphatics are not present in the thy-
roid of Elasmobranch fishes was founded on insufficient evidence
and is incorrect.

5. Lymphatics are present in considerable numbers both in and

208 JEREMIAH S. FERGUSON

about the thyroid gland and can be demonstrated by injection and
in sections; they are true ‘‘ vasa lymphatica.”’

6. The blood-vessels within the thyroid gland terminate in a .
network of ‘‘ venae lymphaticae”’ which invest the follicles, receive
the vasa lymphatica, and transmit either or both blood and lymph,
under varying conditions of blood-pressure.

7. The thyroid artery arises from the ventral end of the efferent
hypobranchial arterial loop contained in the hyoidean hemibranch
and the adjacent half of the first holobranch by an independent
origin or by a common stem with the mandibular or submental
artery.

8. The thyroid veins in these fishes for the most part enter the
‘thyroid sinus,” a mass of veins and lymphatic vessels which pour
their blood into the hyoid sinuses.

9. The rhythmic respiratory movements of the pharyngeal wall
cause the thyroid sinus to act somewhat after the manner of a
‘“‘vein-heart”’: or ‘‘lymph-heart.”’

10. The hypobranchial arterial system is formed as described
by Hyrtl in 1858 and 1872, and the direction of the flow of its
blood is from the gill vessels toward the coronary and other ter-
minal arteries, as indicated by Hyrtl and again by Parker and
Davis in 1899, and not from the subclavian artery toward the
coronaries, as described by T. J. Parker in 1886. The hypobran-
chial artery of T. J. Parker, forming an anastomosis between the
subclavian and median hypobranchial arteries, is of insignificant
importance and is frequently wanting.

11. The relative volume and distribution of the “colloid” in
the glands of different species indicates that this substance is a
retention product, formed from the albuminous secretion of the
follicular epithelium.

12. The further changes occurring in the “colloid” indicate
the possibility of its usefulness as a sort of stored-up secretion.

13. The follicular epithelium contains both “chief” and “‘col-
loid”’ cells, the latter being even more numerous and. characteris-
tic than in the mammalian thyroid.

14. The parenchymal epithelium is in intimate relation with the
vasa and venae lymphaticae; it rests directly upon or in close
proximity to the endothelial wall of these vessels.

THE ANATOMY OF THE THYROID GLAND

209

EXPLANATION OF FIGURES

Abbreviations

I—YJ, first to fifth branchial clefts

A. lat., lateral artery

A. thry., thyroid artery

A. thyr. imp., arteria thyreoidea impar

Art., artery

b. hy., basi-hyal cartilage

cap., fibrous capsule of the thyroid
gland

ch. ep., chief cells of the follicular
epithelium

col., colloid

com., commissural artery

cor., coronary arteries

coraco-mdb., coraco-mandibular artery

coracoid, coracoid artery

D A., dorsal aorta

en., vascular endothelium

ep., epithelium of the thyroid follicles

epibr., epibranchial artery

fol. thyr., follicles of the thyroid gland

gastric, gastric arteries

hy. sn., hyoid sinus

hypobr., hypobranchial artery

hypobr.’, its anastomosis with the com-
missural artery.

L., vasa lymphatica ’

l. c., lateral commissural arteries

Lat. hypobr., lateral hypobranchial ar-
tery

M.c.br., coraco-branchial muscle

M.c. hy., coraco-hyoideus muscle

M. c. mdb., coraco-mandibularis muscle

Med. hypobr., median hypobranchial
artery

p. ¢c., pericardial arteries

pharyn., pharyngeal arteries

R B C, red blood cells.

s. a. v., ventral arterial sinus of the
first hypobranchial loop.

sbel., subclavian artery

thyr., thyroid gland

thyr. sn., thyroid sinus

V. vein

V A, ventral aorta

V L, venae lymphaticae

W B C, white blood cells

z., point of anastomosis of vasa and
venae lymphaticae

y., injected carmine granules in the
venae lymphaticae

210 JEREMIAH §8. FERGUSON

BIBLIOGRAPHY

Baber, E.C. 1881 Phil. Trans., Roy. Soc. London, 172, 577.

Batrour, F. M. 1881 Treatise on Comp. Anat., London 2, 626.

Brivce, T. W. 1904 Cambridge Nat. Hist., London, 7.

Coxe, F. J.. 1905 Anat. Anz., 27, 323.

Dre Meuron, P. 1886 Ree. zool. suisse, 3, 517.

Dourn, A. 1884 Mitth. a. d. zool. Station z. Neapel, 5, 102.

Ecker U. WIEDERSHEIM. 1904 Anat. des Frosches, Braunschweig, 205.

Favaro, 1906 Quoted by Eisler, Schwalbe’s Jahresb., 12, 323.

Comins J. 1896 Thése Paris.

Hyrtu, J. 1858 Denks. d. k. Akad. Wissen., Wien, Math. Nat. Cl., 15, 1.
1872 Denks. d. k. Akad. Wissen., Wien, Math. Nat. Cl., 32, 263.

LaGENDoRF, O. 1889 Arch. f. Physiol., Suppl. Bd., 219.

LomBarpD, G. D. 1909 Biol. Bull., 18, 39.

Masor,-R. H. 1909 Am. J. Anat., 9, 475.

Mavrer, F. 1886 Morph. Jahrb., 11, 129.
1888 Morph. Jahrb., 13, 296.

Miter, W. 1871 Jena. Zeitschr., 6, 428.

ParKER, G. H. anp Davis, F. K. 1899 Proc. Bost. Soc. Nat. Hist., 29, 163.

Parker, T. J. 1880 Trans. and Proc. New Zealand Institute, 13, 413.
1886 Phil. Trans., Roy. Soc. London, 177, 685.

PEREMISCHKO 1867 Zeitschr. f. wis. Zool., 17, 279.

SaBin, F.R. 1909 Am. J. Anat., 9, 43.

ScuaFFER 1906 Anat. Anz:, 28, 65.

Srmon 1844 Phil. Trans., Roy. Soc. London, pt. 1, 295.

StockarD, C. R. 1906 Anat. Anz., 29, 91.

Tscuruwsky 1903 Arch. f. d. ges. Physiol., 97, 210.

TurRNER 1874 J. Anat. and Physiol., 8, 285.

Van BEMMELEN, J. F. 1887 Zool. Anz., 10, 88.

Wacner, R. 1853 Handworterbuch d. Physiol., Braunschweig, 4, 111.

WIEDERSHEIM, R. 1907 Comp. Anat. of the Vertebrates, p. 432.

Reprinted from JouRNAL or MorpHouoey, Vou. 21, No. 4, Supplement,
February, 1911

THE THYREOID GLAND OF THE TELEOSTS

J. F. GUDERNATSCH

From the Department of Embryology and Experimental Morphology,
Cornell University Medical School, New York City

TWENTY-ONE TEXT FIGURES AND FIVE PLATES

During the summer of 1909 at the suggestion of Dr. C. R. Stock-
ard! I undertook the study of the distribution of thyreoid
tissue within the gill region of Teleosts, especially of the trout.
It seemed important to clear up certain doubtful facts in this
connection, since this organ of the trout is liable to disease and at
present is attracting considerable attention in cancer research.
The examination of but a small number of species brought such an
abundance of interesting material to light that I determined to
carry out a comparative study of the anatomy and histology of
the thyreoid gland in a large number of Teleosts, and to summarize
our entire knowledge of the organ in this group of vertebrates.
At the same time I attempted, by comparing the present results
with the facts known of the thyreoid in other classes, to define
more clearly the features of this organ in the entire vertebrate
group.

It gives me pleasure to express my best thanks to The Wistar
Institute of Anatomy and to Prof. F. R. Lille for the use of a
room in the Marine Biological Laboratory at Woods Hole, Mass.
Some of the species were obtained from the New York Aquarium,
for which I wish to thank the Director, Mr. C. H. Townsend.

Twenty families of Teleosts including twenty-nine species were
investigated, the detailed description of which I give in the special

1 My thanks are due to Professor Stockard for many suggestions during the
work and for carefully revising this paper.

JOURNAL OF MORPHOLOGY, VOL. 21, No. 4
SUPPLEMENT: FEBRUARY, 1911

710 J. F. GUDERNATSCH

part of this paper. The results demonstrate the possibility of
unusually wide variation in the thyreoid gland and establish a
continuous series of transitions from one form to the other.

The general part deals with the anatomy, histology and embry-
ology of the organ, and is based on the facts gained from a system-
atic comparison of all the species.

GENERAL PART

ANATOMY OF THE GLAND

The literature relating to the structure and the location of the
thyreoid gland in Teleosts and fishes in general is comparatively
meagre. Long after the presence of this interesting organ had
been known in the higher vertebrates it was also found in fishes.
Simon was the first to demonstrate the existence of a thyreoid
gland in this lowest class of vertebrates, but as he supported his
statements only by macroscopic examination, it is not certain
whether everything he regarded as thyreoid really belongs to this
organ.

The peculiar anatomy of the gland renders it at times very diff-
cult, if not entirely impossible,to diagnose tissues in the gill region
as thyreoid without a microscopic examination. I have several
times, especially when particles of the thyreoid were diffusely
scattered, regarded small masses of tissue as thyreoid which under
the microscope did not prove to be such. Simon’s reports on the
different locations of the gland are, therefore, not to be relied
upon and it is for the above reason especially that his discoveries
in the Gadidae, Cyprinus, Anableps, Esox and Exocoetus are to be
very strongly doubted. In these species he locates the thyreoid
gland far dorsally in the region of the soft palate. Maurer has
since demonstrated the true position of the thyreoid in the carp
at least, and although Simon’s statements have not been contra-
dicted for the other species, they seem to be, from the phylo-
genetic point of view, entirely untenable. In some other species,
however, Merlangus, Anguilla etc., Simon appears to have found
the organ in his dissections, as he places it near the bifurcation

THYREOID GLAND OF THE TELEOSTS 711

of the first gill arteries. It seems strange, on the other hand, that
he was unable to locate the thyreoid gland in some other fish,
among them those in which it is easily visible to the naked
eye. Thus he failed to see it in Perca, Mugil, Trigla, Scom-
ber, Tinca, Salmo, (Salmo fario), Clupea, Pleuronectes, Hippo-
glossus, Rhombus, Solea, Cyclopterus, Gymnotus and Balistes.
In all these species, as far as they were at my disposal, I could
demonstrate the thyreoid gland as a well developed organ, and feel
certain that it is also present in the others. There seems to be
little, if any doubt that the thyreoid gland is an organ belonging
without exception to all fishes and vertebrates in general.

Of the later investigations describing the thyreoid gland in
Teleosts, Baber’s work (1881) should be mentioned, although he
studied the gland of only one species, the conger eel. A later
paper by Maurer (1886) deals principally with the embryology of
this organ in the trout, but also contains some remarks on its
comparative anatomy.

The thyreoid gland of the vertebrates, as is well known, is
closely connected in its development with the median ventral
wall of the pharynx and the gill arches. These relationships are
only slightly different whether the gills persist throughout life
as in the fishes, or are found only in larval stages asin the Amphibia,
or finally transform in later embryonic life as is the case in the
Amniota. The location of the thyreoid in the animal body is
therefore dependent upon the gill region. While in the higher
vertebrates the topographical arrangements become more or less
changed or rendered indistinct during the progress of development
we find them in the fishes typically marked.

The region of distribution of the thyreoid gland in the fish
extends below the floor of the pharynx, in the body of the tongue,
between the gill arches and back posteriorly behind the origin of
the third and fourth branchial arteries from the ventral aorta.
Maurer limits the region of distribution anteriorly by the bifur-
cation of the aorta and posteriorly by the last branchial aortic
arch, but these lines are certainly too limited, especially in the
anterior direction. In some species the main body lies in front
of the aortic bifurcation. The posterior limit, however, only

712 J. F. GUDERNATSCH

exceptionally occurs behind the last branchial arch. The single
parts of the basibranchiale define this ‘thyreoid region’ dorsally,
while ventrally the paired musculus sternohyoideus is spread out
beneath the organ. This region is at the same time that of the
ventral aorta and its branches to each of the gills.

Thus the thyreoid gland is located along the trunk through which
the blood for the entire body is pumped from the heart into the
respiratory organs. The narrow cleft between the bony parts of
the floor of the pharynx dorsally and the muscles ventrally is
completely filled with thyreoid tissue except for the space occupied
by the large arterial trunks. This region, as we see from the
extensive literature on the visceral skeleton and musculature in
fish, in the manifold development of its bony (cartilaginous) and
muscular parts shows a decided tendency to vary. It is only
natural that this tendency should be found in the thyreoid gland
also; since it has to accommodate itself to the configuration of
the tissues just mentioned, a pronounced adaptability must be
of the greatest benefit to it. The property of variation is possessed
by the thyreoid gland of the Teleosts to a most striking degree
and within thesamespeciesratherremarkable differences are found.
Twelve weak-fish (Cynoscion) for instance, all differed in the exten-
sion and position of their respective thyreoids. Similar conditions
were observed in other species. This variability within the species
may indicate that in the thyreoid gland we have a very unstable
organ, which perhaps in vertebrate phylogeny has not yet acquired
its final condition. We know that in the higher classes of verte-
brates there is the same variability among individuals regarding
the development of their thyreoid glands. In mammalia the
individual variation is very great. The lobes may have different
forms, and give to the organ a paired appearance, or there may
be a more or less well developed isthmus between them. Interest-
ing comparisons have been made especially in man.? In the
phylogenetically younger epithelial bodies the variation is still
larger. All of these facts indicate that the gill slits and their

* Marshall (’95) examined the thyreoid glands in sixty children in which he found
all possible variations.

THYREOID GLAND OF THE TELEOSTS fis

derivates are still easily modifiable and do not yet represent a per-
manent condition.

The thyreoid gland of Teleosts is not a single compact organ,
as we find it in the higher vertebrates, where the small parts of the
gland, the follicles, are united into one complex and enclosed by a
common capsule of connective tissue. Only in such a case would
the term ‘gland’ be justified, since here numerous anatomical
elements possessing the same physiological function are closely
connected. The Teleosts, however, possess numerous elements,
whose totality from a physiological standpoint one must regard
as a thyreoid gland, while anatomically we are unable sharply
to define the organ in question in this group of vertebrates. In
most cases we can speak of thyreoid follicles only, or groups of
follicles, in pointing out the distribution of the organ. Thus in
plates I and II, not the thyreoid glands of the respective species,
but the regions of distribution of the thyreoid follicles themselves
are figured. On plate III, however, which indicates the variation
in the location of the gland within the species, real parts of the
thyreoid are indicated, so far as they were macroscopically visible.

In the more closely defined region we may find thyreoid tissue
in all parts. It is usually of a brownish yellow color. ‘The follicles
are. generally most densely located in the neighborhood of the
ventral aorta or of the branchial arteries. Those places, particu-
larly, are favored where the branchial arches arise from the aortic
stem. Follicles are most abundant at the origin of the second
gill arteries, that of the first being next, and finally the roots of
the last branches have fewest thyreoid follicles about them. A
more or less dense accumulation is always found along the stem
of the ventral aorta, which may be completely surrounded by
thyreoid tissue. In other cases dense accumulations of follicles
are located either dorsally or ventrally to the aorta.

The glandular tissue is usually distributed so that it is more
densely accumulated in the central region, while towards the pe-
riphery a more and more pronounced dissolution and scattering of
the follicles takes place. These conditions cannot, however, be
strictly generalized, as we find cases in which, even in the most
central portions, the follicles are not more closely arranged than

714 J. F. GUDERNATSCH

in the peripheral. In some instances, as in Cynoscion, rather well
developed central portions are found which can be recognized by
the naked eye. But even in these cases numerous follicles lie well
separated from the main body. Thus we have all transitional
stages from a perfect dispersion of the follicles to a rather compact
union of them. It is possible that further investigations may
show cases in which the organ is still more compact than in those
thus far examined, and so present a structure similar to that in
higher vertebrates. Judging from my observations, however,
this does not seem probable, and at present I am inclined to re-
gard the conditions found in Sarda (ede Special Part) as the limit
of compactness in the series.

The cephalad and caudad extensions of the thyreoid gland vary
very much. In general, it might be said that a spreading out to-
ward the tip of the tongue takes place in all cases, while towards
the heart the distribution is not so uniform. Far cephalad of the
first aortic bifurcation we find single follicles scattered below the
hyoid bones. The caudal limit of the thyreoid gland usually lies
between the second and third aortic branches, or at the third.
Rarely does it go beyond this point, and if so, with a few excep-
tions, only scattered follicles are found in the posterior region.
Thus we find an accumulation of the glandular elements around
the anterior part of the ventral aorta, with follicles scattered
towards the head and the heart.

The organ decreases in mass in an anterior-posterior direction.
In one instance, Siphostoma, just the reverse is the case.

The embryonic center from which the thyreoid starts to grow
lies between the first and second gill branches, this place in the
adult animal is near the aortic bifurcation, and in many cases
we find the main part of the organ in this region; while in other
cases, Cynoscion and Tautogolabrus, it is exclusively there.
From the region of the aortic bifurcation the thyreoid elements
travel so as to occupy the different positions which are described
in the special part of this paper. The migration of thyreoid
follicles occurs more particularly in the direction of the heart,
although a cephalad migration is decidedly pronounced. The
development of the hyoid bones obstructs the anterior spreading

THYREOID GLAND OF THE TELEOSTS 715

of the follicles to some extent. The follicles apparently tend to
go around the basihyale and along its sides towards the tip of the
tongue. In the dogfish and shark where the hyoid region offers
a comparatively free space we find it occupied by the conipact
thyreoid, which is pushed slightly forward of the aortic bifurcation
(Ferguson). In these animals also the thyreoid is originally placed
in the bifurcation of the truncus; later, according to W. Miiller,
the anlage moves forward and becomes encapsuled. In Raja,
on the other hand, it remains in the bifurcation.

The dorso-ventral, in combination with the lateral, extension
of the thyreoid follicles seems to be more dependent upon the con-
figuration of the pharyngeal floor than does the cephalo-caudad
extension. In fishes, in which the isthmus region is deep and
narrow, as in Brevoortia, we find, as might be expected, the dorso-
ventral distribution of follicles far surpassing the lateral. While
in other species, for instance, Tautoga, in which the floor of the
pharynx is very broad, the lateral extension is the important one.
In general it may be said that the lateral outweighs the dorso-
ventral distribution of follicles.

Dorsally the follicles are usually found between the ventral
aorta and the copulae of the gill arches. Incases where those skele-
tal parts come close to the vessel the follicles are forced away
laterally, and sometimes intrude into the spaces between the copu-
lae and hypobranchialia. When the parts of the basibranchiale
lie well separated the follicles extend up between the copulae and
come to le close to the mucous membrane of the pharyngeal
floor.

The main mass of the organ lies almost exclusively above, or
dorsal, to the ventral aorta. This is opposed to Maurer’s state-
ment of the case. Below the aorta is usually found the smaller
part of the gland and the follicles are also more loosely scattered.
The development of the thyreoid gland above the aorta should
be expected since there is usually much more open space between
the aorta and the gill arches than is found below the aorta where
the muscles lie close to the vessels. The thyreoid elements en-
deavor to intrude below the aorta as much as possible, and when
this vessel, in the region of the third branchial arteries, sinks deeper

716 J. F. GUDERNATSCH

into the musculus sternohyoideus, the follicles follow in the course
and are thus distributed far ventrally within the muscle. The
number of follicles along the sides of the aorta is always less than
either above or belowit. This by no means contradicts the state-
ment made above that the lateral ex’ension of follicles outweighs
the dorso-ventral one. Only in the genus Fundulus, especially
in majalis, less so in heteroclitus, where a transverse inter-
branchial muscle pushes the vessels away from the skeletal parts,
is the dorsal extension small, or sometimes lacking entirely. Here
the follicles extend directly away from the aorta towards the
bases of the gills.

Along the aortic stem between the bases of the gill arteries
the lateral extension is somewhat limited and reaches its height
along the branchial arteries. The vessels seem to serve as bases
along which the follicles migrate. The free space about the gill
arteries becomes narrower and narrower as the gill arches are
approached and therefore the number and size of the follicles
decrease towards these points until there is no more room for
extension. When, however, there exists an especially open passage
along the vessels the follicles may even extend into the gill arches,
to a considerable distance beyond the point of their origin. Such
cases are common in trout (text fig. 7D).

The peculiar distribution of the thyreoid elements forces us
to regard the organ in bony fishes as unpaired, a view also sup-
ported by embryology. This statement should be especially
emphasized, as in Wiedersheim’s Comparative Anatomy, 1907,
the author still speaks of the thyreoid gland in the Teleosts as a
paired organ, although Maurer in 1886 (p. 134) criticizes this
statement in an earlier edition. On another page, (p. 140) Maurer
himself claims that the main bulk of the organ at a certain stage
is not paired, while later single portions of the gland lying in the
median line, as well as on both sides of the truncus arteriosus,
take a paired arrangement. This is certainly incorrect, since the
follicle groups on the sides of the aorta are not only unpaired but
are also not bilaterally arranged.

The relationship between the thyreoid gland and the stem of the
ventral aorta is purely anatomical and without any physiological

THYREOID GLAND OF THE TELEOSTS ray;

importance. The thyreoid does not receive its blood supply from
this group of vessels, since they carry only venous blood, and the
arterial blood which nourishes the gland comes from a special
thyreoid artery. In Petromyzon, however, Cori claims that the
arteria thyreoidea arises from the truncus arteriosus; this is prob-
ably an error, as he also finds the ventral carotid connected with
the truncus. The thyreoid artery, as Silvester demonstrated in
Lopholatilus and twenty other species of Teleosts by his perfected
method of injection, arises as a dorsal branch from the united
right and left fourth commissural arteries. The latter vessels
originate from the second efferent branchial arteries and unite in
the median line below the thyreoid and the aorta as the hypo-
branchial artery. Shortly after the union of the fourth commis-
sural arteries the thyreoid artery branches off from the dorsal
side and immediately enters the gland in its posterior region.
Whether the widely scattered follicles all receive their capillaries
from this one vessel cannot at present be stated, though it would
seem very doubtful especially in the case of the more anteriorly
isolated follicles.

In Selachians, where the thyreoid is pushed far forward, the
arteria thyreo-spiracularis (Dohrn) originates in the first aortic
arch from the arteriae efferentes of the hyoid gill. In Teleosts,
also, the first aortic arch breaks up into a capillary network.
Dohrn, therefore, speaks of an arteria thyreo-spiracularis. Per-
haps it is from this vessel that the most cephalad parts of the
thyreoid gland receive their blood supply. The artery pointed
out by Silvester seems, however, to supply the bulk of the organ,
and the term arteria thyreoidea as applied to it is apparently
justified.

It is of interest to recall Simon’s statement, which was also
supported by others, that the thyreoid gland is placed in the blood
system so as to regulate the supply of blood to the brain. This,
in a way, was a foreshadowing of our present views that the phy-
siological action of the thyreoid gland exerts an important influence
on the central nervous system.

The venous blood from the thyreoid gland passes into the thy-
reoid vein, a vessel, which also collects the veins from the muscula-

718 J. F. GUDERNATSCH

ture below the aorta and carries the blood directly into the sinus
venosus.

Little is known about the relation of the thyreoid gland to the
lymph system. This is largely due to the fact that in the fishes
the lymph vessels are in a much closer connection with the venous
system than in the higher vertebrates. It is almost impossible
to distinguish between veins and lymphatics by the injection
method.

In many species large cavities lie around the aorta, two dorsal
ones being constant in trout. These are extraordinarily large and
lined with endothelium and although they often contain blood
corpuscles there is little doubt that they are lymph sinuses. The
corpuscles probably come in from the venous system, or possibly
by traumatic haemorrhages. A further fact in favor of their
being lymph sinuses is that no descriptions of large veins in this
region has come from the numerous injections of the circulatory
system of Teleosts. In some species there is only one large ‘lymph
sinus’ which surrounds the aorta dorsally and laterally.

- DEVELOPMENT OF THE THYREOID

Only one contribution deals with the embryology of the thy-
reoid in Teleosts, this is by Maurer (’86) who traces its develop-
ment in the brcok trout. The gland arises in much the same
manner as it does in the other classes of vertebrates.

The thyreoid develops very early in the Teleosts, after the
first gill slit has broken through,? as an unpaired evagination of
the stratified epithelium on the ventral side of the pharynx between
the first andsecond gillpockets. Itisthus placed in the curve of the
S-like tubular heart before the gill arteries have developed, with the
exception of that to the hyoid arch. The vesicular thyreoid anlage
very soon separates itself from the pharynx and enlarges by bud-
ding. The organ lies close to the tubular heart, but only remains
for a short time near the place of its origin. With the development

* In Hertwig’s Handbuch d. Entwicklungslehre II, 1, 1906, Maurer states, how-
ever, that the anlage of the thyreoid appears in all Gnathostoma before the break-
ing through of the first gill slits.

THYREOID GLAND OF THE TELEOSTS 719

and shifting of the heart and aorta as well as by its own growth
the thyreoid gland comes to lie far from its original position. The
absence of a capsule of connective tissue similar to that in higher
vertebrates admits the loosening and separation of the thyreoid
follicles in the bony-fish.+‘

The Teleosts show a condition of the thyreoid gland somewhat
similar to that in Myxine glutinosa, as W. Miiller, Cole,
Schaeffer and Maurerstate. The folliclesin Myxine, partly isolated,
partly in groups, are found between the pharynx and the truncus
arteriosus throughout the gill region. In the Teleosts, how-
ever, the gland also extends below the truncus. In the skate
Baber observes ‘‘a single body and a few detached vesicles’’;
in the Amphibians separated particles have also been described.
Yet in bothof these groups the thyreoid possesses a capsule, which
sends septa into the inner portions, as W. Miiller has shown for
Acanthias and Raja. Maurer finds a delicate connective tissue
capsule in the Urodela. These observations on Selachii and Am-
phibia, however, are exceptional and the small detached particles
can only be looked upon as ‘aberrant thyreoids’ the main thyreoid
in all cases being a sharply defined body. Maurer observed in the
Urodela that a breaking up of the thyreoid into smallerparts
occasionally occurred. These accessory thyreoid glands were
parts of the former isthmus which, after the anlage had divided,
persisted and remained in their original position, while the true
halves moved in a postero-ventral direction. In the Ophidia the
organ is compact and encapsuled; but in the Saurii, according to
W. Miler, the interstitial tissue increases so much through the
accumulation of fat, that the glandular tissue proper is broken
up into irregular groups which are sometimes completely discon-
nected. We have here a dissolution within the capsule suggesting
that the connective tissue capsule is the only factor in other verte-
brates which prevents the thyreoid elements from becoming
scattered about as they are in Myxinoids and Teleosts.

4 The elements of the Teleost pancreas are similarly scattered in the mesen-
terium.

720 J. F. GUDERNATSCH

During development of the Teleosts some follicles cling to the
wall of the aorta and are in later life found along it. Usually
the follicles become arranged into several distinct groups, forming
different centers of growth, as is shown by Cynoscion in plate
III. With the branching off of the gill arteries from the aorta
thyreoid material is carried out laterally towards the gills and
spreads in this region. This accounts for the larger lateral exten-
sion of thyreoid follicles along the gill vessels rather than in inter-
mediate regions. The larger vessels form a substratum upon which
the follicles migrate as do also the smaller vessels and especially
the lymphatic vessels. The larger vessels are means for the an-
tero-posterior dispersion while the smaller ones allow the migra-
tion of follicles from the denser central thyreoid portions towards
the periphery. Even the most peripheral follicles are usually
found near blood capillaries although they do not necessarily
come in close contact with them. The way in which these isolated
follicles function is not clear. They certainly seem normal! and
contain colloid.

The growth of the connective tissue and fat in which the folli-
cles are imbedded favors their dispersion from the central portions;
thus a combination of influences are at work to widen the thyreoid
region as much as possible. W. Miller regards the immense
development of the ‘interstitial’ tissue as alone responsible for
the dissolution of the thyreoid into isolated groups. He no doubt
refers to fat and connective tissue, as we shall see below that the
term ‘interstitial’ is not properly used in this case. Although
the growth of these tissues may be an important factor I donot
regard it as primary, since in the first place, even in young indi-
viduals, the follicles are found isolated, and secondly, the breaking
apart of a formerly compact organ by excessive growth of connec-
tive tissue would certainly not account for the carrying of the
follicles into the muscles and gills.

The follicles actually seem to overcome the obstruction offered
by other tissues in their course and may even penetrate into them.
In trout and Micropogon the thyreoid follicles are at times im-
bedded in the muscle tissue, into which they creep between the
connective tissue lamellae or along the blood vessels. Realactiv-

THYREOID GLAND OF THE TELEOSTS 721

ity on the part of the follicles is most unlikely, and the probability
is that they are simply passively pushed or pulled as circum-
stances may have it. The forces in development unite to make it
possible for the thyreoid gland to spread, and so form a greater
amount of functional tissue than could be contained in a compact
organ situated in the narrow space between the basihyale and the
ventral musculature.

Little is known of the manner in which the thyreoid gland grows
and forms new follicles, and contradictory statements are also
found in the literature regarding the primary anlage of the organ.
It is scarcely conceivable that a vesicular anlage shopld exist in
all fishes except Ceratodus in which Greil observed a solid one.
Before the solid outpushing in Ceratodus separates from the
pharyngeal wall it is said to become vesicular, a process exactly
the contrary to the usual one.

Amphibians are believed to have a solid bud-like thyreoid
anlage. Maurer states that two days after its evagination
the thyreoid is solid in the Anura, and W. Miiller observed a solid
anlage in Rana temporaria and platyrrhinus, in which the first
lumen appeared in 25 mm. larvae, after the gland had divided
into two halves. In the Urodela Maurer records a solid epithelial
bud, Livini finds the same in Salamandrina perspicillata and
Muthmann in Triton alpestris. Platt claims that Maurer’s
description does not apply in all the Urodela, as is shown by the
condition in Necturus.

The reptiles, birds and mammals are said by the majority of
observers to show a vesicular thyreoid anlage, which changes into
a compact organ from which follicles later originate. Kolliker,
however, observed in the rabbit a thickening in the ventral wall
_of the pharynx, from which a wart-like solid process was cut off.
Born also records the same for the pig. (Both authors quoted from
Streckeisen).

This point is of importance in phylogenetic interpretations
since our present views regarding the ancestry of the thyreoid
gland are mainly based on a similar evagination, that for the endo-
style, found in the Tunicates and Amphioxus.

Ta. J. F. GUDERNATSCH

Maurer finds in the trout a primarily globular vesicle stretch-
ing in an antero-posterior direction, andon the 41st day of develop-
ment lying ventral to the stem of the aorta. If these statements
apply to all Teleosts, the thyreoid must first originate dorsal to
the aorta and early migrate ventrally and later return to a position
dorsal to the vessel, since it usually occurs there in the adult. The
condition in the Teleosts is similar to that in the Myxinoids,
where Stockard describes the origin of the thyreoid as a median
down-pushing from the ventral floor of the pharynx throughout
the entire gill area, and consisting, in newly hatched Bdello-
stoma, of diffusely scattered alveoli below the pharynx and
above the median branchial artery (ventral aorta).

In the trout, where development is rather slow, Maurer observes
that 35 days after fertilization, when the embryo is about 6 mm.
long the first thyreoid vesicle begins to pinch away from the phar-
ynx. While originally the evagination, visible on the 28th day,
possesses a stratified epithelium, it has on the 35th day a single
layer of cuboidal cells. Three weeks later the whole stem of the
aorta is surrounded by follicles. I find in rainbow-trout, one
month old, or only 30 days older than those mentioned by Maurer,
that the majority of follicles, and the larger ones, lie above the
aorta.

Maurer also observes that in the brook-trout shortly after the
first follicles have appeared the organ grows so rapidly that
for a considerable period it surrounds the aorta as a compact °
mass. “In very late embryos, the growth of the thyreoid does not
keep pace with that of the artery; thus the gland breaks away
from the aorta and separates into a number of irregular clusters of
different sizes lying either laterally partly paired or dorsal or
ventral to the aorta, always, however, in its immediate neigh-
borhood.”’ He records the main mass of the gland in trout of
even 25 cm. as being compact and situated ventrally between the
second and third branchial arteries, and it is only in animals of
30-40 cm. that the thyreoid breaks up into the clusters of follicles
characteristic of the adult. Maurer describes the same conditions
in a number of other species, of which only the eel was at my
disposal. The age of the eel I examined was unknown, though

THYREOID GLAND OF THE TELEOSTS 723

according to Maurer it must have been rather old, yet it measured
only 30 cm. long.

Maurer’s observations do not accord with the conditions I
find in rainbow-trout four weeks old, nor in 25-30 cm. brook trout.
As before stated, there is no paired arrangement of the thyreoid
clusters, and the follicles are also in many cases distantly removed
from the stem of the aorta. Other differences may be either
due to specific or individual variations. Maurer’s statements
would indicate that the thyreoid gland tends to preserve its origi-
nal unity, being finally broken up by force. My observation, how-
ever, seems to show the contrary, at least inSalmo irideus and fonti-
nalis. In individuals one year old the follicles are more densely
packed than in those one month old, although the intervening
spaces have grown larger. The follicles also have become more
numerous. This seems to warrant the supposition, that the thy-
reoid elements are disassociated at an early stage and subsequently
multiply.

The multiplication of the follicles is described by Maurer as
being very simple. While the epithelial cells are increasing in
number after the forty-first day (in trout) solid buds appear on
the primary vesicle, which very soon form central cavities and
then pinch away. We do not know whether a similar process is
maintained in later life, follicles coming from follicles, or whether
new follicles are derived only from primary epithelial cells multi-
plying and forming a lumen. The latter supposition would more
readily explain the scattering of thyreoid elements, germ elements
I might say, to distant regions. L. Miiller believes the new folli-
cles to originate from old ones by buds from the epithelium which
are subsequently pinched off. Baber contributes an interesting
observation in the conger eel where in the wall of large follicles
small ones sometimes lie imbedded so deeply that the epithelium
between them is flattened out. Baber thinks that at times the
wall breaks through and the two lumina are united. In other
cases, however, the small imbedded follicles grow out and become
independent.

The epithelial tubes found in the thyreoid of higher vertebrates
as transitory growth stages are absent in the Teleosts. In the

724 J. F. GUDERNATSCH

Urodela a solid cylinder of epithelial cells from which the follicles
pinch away, exists for from two to four weeks. Thesecylinders are
observed in sheep and pig embryos up to the 20 em. embryos and
in man up to the 24cm. embryo. The follicles in fish are formed
comparatively earlier, and perhaps the gland functions earlier.

Thus a rapid multiplication of follicles occurs in the Teleosts
without the formation of cell cylinders. This is an exception to
W. Miiller’s claim that the thyreoid gland inall vertebrates passes
through three stages: (1) a severing of the anlage from the pharynx;
(2) formation of a network of tubes of glandular epithelium;
_ and (3) the formation of follicles from these tubes. In the Teleosts
and also in the Myxinoids, as Stockard has shown for Bdellos-
toma, the second stage seems to be suppressed or absent.

The first appearance of colloid in the thyreoid gland is generally
thought to occur early in lower vertebrates but very late in the
higher ones, towards the end of fetal life or often not until extra-
uterine life.

Maurer reports colloid in the trout thyreoid on the forty-first
day of embryonic development. How far this early appearance of
colloid is connected with the function of the organ is unknown.
From a comparative physiological standpoint it would seem that
in the lower forms the thyreoid might function much earlier than
in the Placentalia, where in intra-uterine life the gland of the
mother might supply the needs of the developing embryo.®

HISTOLOGY OF THE GLAND

The histological structure of the thyreoid gland in Teleosts has
been little studied. The meagre observations made by Baber in
1881, describing some features of the thyreoid in the conger eel
were the first reported. Maurer later (’86) mentions a few points
regarding the histology of the thyreoid in the trout and carp.

The microscopic appearance of the gland varies as much as does
its anatomical structure. In sections from some specimens the

° In young mammalian embryos Peremeschko found no colloid, in older embryos
it occasionally existed, while in young animals colloid was present in the majority
of follicles and in old ones in all of them.

THYREOID GLAND OF THE TELEOSTS 725

follicles are closely arranged and so densely packed that apparently
only lymph spaces exist between them, in others we find the folli-
cles more loosely connected and suspended in the connective tis-
sue; while again in other specimens they lie so far apart that they
can scarcely be thought of as belonging to one organ. The his-
tological appearances also differ very much within the individual,
depending upon the region from which the section is taken.

When the arrangement issuch that the thyreoid may be dissected
out and then sectioned, the follicles are found to be rather densely
packed (pl. V, fig. 17). By this method, however, we are unable
to get a correct idea of the extension of the thyreoid and the ar-
rangement of its follicles, since it is only possible to remove the
somewhat denser masses around the stem of the aorta, usually near
the base of the second aortic arch, and all the particles in front
and behind this region still remain. Properly to study the general
distribution of the thyreoid follicles serial sections through the
entire gill region are absolutely essential.

The spaces between the muscles, branchial arteries and gill
arches are filled by wide-meshed connective and fatty tissue. In
these tissues the follicles are suspended. The connective tissue
is, therefore, not so directly a part of the thyreoid organ inthese
fish as it is in the encapsuled organ of mammals. The primary
object of this tissue is to form a connection between the muscles
and bones without regard to whether there may be thyreoid
tissue in the region or not. True interstitial tissue, as such, is
not found in this diffusely scattered thyreoid organ. Of course,
the tissue in which the follicles lie imbedded performs the same
function as does the capsule in higher vertebrates: in both cases
it serves to support the follicles. In glands, where many follicles
are accumulated in one mass, as in Cynoscion, or in the central
portions of some others, for instance the trout, the supporting
tissue may be regarded as part of those masses, but not as part of
the entire thyreoid gland; here also the formation of connective
tissue is the primary process, and the suspending of the follicles
only a secondary one.

The supporting tissue is simple except in two species, Salveli-
nus and Sarda (pl. V, fig. 21) where smooth muscle fibres are freely

JOURNAL OF MORPHOLOGY, VOL, 21, No. 4

726 J. F. GUDERNATSCH

suspended in the connective tissue. These muscle fibres are found
especially below the aorta, where they approach the follicles and
at times surroundthem. Thisis accomplished by the fibrillae of a
bundle loosening up a little, then enclosing a row of follicles and
finally uniting again.®

Regarding the number, size and form of the follicles, all varia-
tions exist which have been demonstrated by comparative inves-
tigations in the other classes of vertebrates. The size of the fol-
licles is, in general, in reverse proportion to theirnumber. The
size, however, is not of great importance, since the chief factor
in the activity of the gland is the epithelial surface; this will be
the larger, the greater the number of small follicles contained in a
given region. Biometric calculations would be interesting in this
direction as experiments have shown that the functional value
of the thyreoid gland varies with the individual. Glands are
found in which the size of the follicles is uniform; in such cases the
follicles are usually large. As a rule, however, the follicles are of
various sizes as would be expected in view of the process of forma-
tion of new follicles. In many cases I have observed that a few
(three or four) follicles are unusually large.

The follicles lying in the central parts are generally larger than
those towards the periphery. This seems quite natural in view of
the mode of extension of the gland. In only one case, Sarda, do
the central portions consist of nests of numerous small follicles
while larger follicles lie peripherally (text fig. 12). This con-
dition resembles somewhat that in birds (Baber), and mammals
(Anderson, Forsyth) and if it be due to the fact that in Sarda the
gland is almost as compact (of course without a capsule) as in the
higher vertebrates (with capsule) then we must suppose that in
such a case new follicles are formed in the centre and are pressed
out towards the periphery, while in the breaking up of the gland
minute parts are continually carried towards the periphery and
there form new follicles. In the first case the peripheral follicles
would be the oldest and in the second the youngest ones.

° Streiff finds muscles between the glandular tissue in the thyreoid of the cat,

Zielinska in a young dog, Wélffler in a child (cit. from L. Miller), L. Miller in an
adult woman. The muscle must have migrated into the gland during the first half

of the embryonic period, before the capsule was formed.

THYREOID GLAND OF THE TELEOSTS tot

The form of the follicles is also variable, most typical perhaps
are the globular or elliptical and tubular types. The smaller
follicles are nearly always circular in section (pl. V, figs. 10-12),
especially when they are free. The shape of the more-closely
packed follicles is influenced by pressure, and may be flat, in-
dented, or irregular in outline. When the follicles lie next to the
cartilages or muscles they are usually oblong-oval, with the longer
side towards the tissue. Single follicles lying in the supporting
tissue, if large, are rarely prefectly circular, but have irregular
outlines due to pressure from the fibres of the substratum. The
shape of these follicles indicates the existence of actual pulling
forces in the supporting tissue.

Not only small irregularities are found in the surface of the
follicles, but also deep invaginations of the epithelium as well
as long evaginations. The follicle may consist of a central body
with sprouts or branches of cylindrical and globular shapes (pl.
V, figs. 15, 16). How far these irregularities in form are connected
with the cutting off of smaller follicles from larger ones could not
be determined. Anderson doubts the multiplication of follicles
by such a process.

It is now generally accepted that no communication from follicle
to follicle exists; the follicles are closed on all sides and perfectly
separated from each other. Sometimes, however, as many as five
follicles are observed in a section, apparently perfectly separated,
but on tracing through the series of sections they all unite into
one follicle (pl. V, fig. 16). This is due to evaginations from
the follicular wall somewhat like the fingers of a glove, which when
cut across, give the appearance of several independent follicles,
while in reality there is only one lumen. In Anguilla chrysopa,
however, there really seemed to be a communicating duct between
two follicles; the lumen of the tube was much narrower than that
of the follicles and the epithelial cells of it were much higher
(pl. V, fig. 15). This closely resembles a ‘Schaltstiick’ as seen in
other glands. This was not due to a waist-like constriction of the
epithelium, but to a far reaching evagination from one follicle with
a globular swelling on the free end representing a second follicle.
There was no colloidal substance in the ‘interealary’ duct.

728 J. F. GUDERNATSCH

Branched follicles are particularly abundant in some species.
In Muraenoides all follicles seem to branch. Baber states that
in young animals the follicles are much more ramified than in
older ones; he, therefore, regards this branching as the method of
follicle multiplication. Anderson, on the other hand, holds the
‘melting’ of the epithelium (a process about which I shall speak
later) at the point where two follicles meet responsible for the
communication between several lumina; this of course is an oppo-
site process from that of budding. Anderson, therefore, believes
that in old animals there are more irregular follicles than inthe
young. It seems to me that the ramification of the folliclesdoes
not depend so largely upon age, but rather on the species.

The follicular epithelium varies but little with the species,
perhaps the number of cells may differ in follicles of the same capa-
city. The epithelium is of the form usually found in the thyreoid
glands of higher vertebrates. All transitions exist from a pave-
ment epithelium of very low broad cells, through cuboidal cells
as high as broad, to very high and narrow cylindrical cells (pl.
V, fig. 10).

The form of the epithelium is probably connected with the age
of the specimen, as it undoubtedly flattens with increasing age.
(In very old human subjects only perfectly flat cells have been
found.) Age, however, can scarcely be the only factor, as in some
species different forms of epitheliumappearat the same time. This
may be due to the different ages of the follicles, though it cannot
be regarded as an absolute rule that the older follicles have a lower
epithelium than the younger. MHiirthle definitely states that
these two factors are independent of one another. Langendorff
points out that the follicles increase in size, not by a flattening
out of the epithelium, but by multiplication of cells. I should
say that both processes may be simultaneously involved since we
often find large follicles with high epithelium, yet karyokinesis
is rarely observed in the epithelial cells. The latter fact led
Stockard to suppose that amitotic cell division might occur in the
growing thyreoid tissue of Bdellostoma.

The different types of epithelium might be accounted for in
still another way by supposing the follicles to be in different

THYREOID GLAND OF THE TELEOSTS 729

stages of activity. Here again we meet with difficulties since the
same follicle sometimes shows high cylindrical epithelium on
one side and a flattened epithelium on the other (pl. V, fig. 11).
Hiurthle considers the flattening and stretching of the ¢ells to be
the final stage in the process of colloid formation. He finds this
type of cells not sporadically, but always in the larger groups of
follicles. The low epithelial cells are still alive and according to
Hirthle may again transform into high ones. Biondi claims that
when the follicle has reached a certain size, the epithelium par-
tially flattens and vanishes, thus establishing a communication
between the follicular lumen and the lymph spaces and allowing
the colloidal material to be poured into the lymph system. The
emptied follicle is said to collapse and from its cell mass a new
follicle originates. Anderson, also, thinks that by a ‘melting’ of
the epithelium a connection is formed between the lymph space
and follicle, but the individual follicle is not destroyed.

In the conger eel Baber finds oval cells between the cylindrical
ones and attributes to them the formation of new follicles, an
idea which I think is incorrect. The two classes of cells could not
be found in the common eel. Baber also finds in the conger eel
‘club-shaped’ cells between the epithelial cells. They are much
narrower than other cells and possess elongated nuclei. Their
free ends project above the general surface and are expanded
‘fan-like;’ the bases may also show a similar condition. Baber
regards them as branched cells, often existing in pairs, and form-
ing stomata which play an important part in absorption and secre-
tion. If this be true such cells should exist in all glands. I was
unable to find these and think perhaps they may have been con-
sequences of his alcohol preservation.

The form of the nucleus changes with the form of the epithe-
lial cell. It is usually circular or somewhat oval in cross section.
When the cell is either cylindrical or flattened, the nucleus becomes
more and more elliptical in shape, with its long axis parallel to
that of the cell. Thus in the first case the long axis of the nucteus
is vertical to the free surface of the cell and in the second parallel
with it. When the nuclei are oblong in spite of the cells being
broad and cubical (for instance in some follicles of Muraneoides)

730 J. F. GUDERNATSCH

they always present the long side to the free cell surface. Fre-
quently narrow cells with oblong nuclei are seen between the
cuboidal cells.

In trout degenerating epithelial cells of small size were observed
with compact nuclei, deeply staining or pyknotie.

The nucleus usually lies at the base of the cell (pl. V, fig. 11)
but may sometimes, especially in an epithelium with many cells,
move a little towards the lumen (pl. V, fig. 10). Nuclei may lie
at different altitudes, in an alternating fashion. One or two
nucleoli are visible.

The shapes of the nuclei usually give no indication of the state
of activity of the cell as Anderson has claimed. Even pyknotic
nuclei usually have regular outlines. An exception to this is seen
in the trout where often, in some varieties almost exclusively,
epithelial cells show nuclei of very irregular shapes, as indicated in
pl. V, fig. 10. .The nuclei are elongated with more or less bent
corners—horse-shoe shape. These were generally found in lower
cells; they may have been degenerating, since they did not stain
as deeply as the normal ones in other parts of the gland, when such
were present. It seems, however, scarcely conceivable that the
epithelium of the entire gland should degenerate, unless from
some pathological condition. (These animals were all reared in
the N. Y. Aquarium.)

The cytoplasm of the cell appears granular and sometimes
stains slightly darker in the basal region. There is no cuticle
lining the lumen, but the refraction of light in this region has
misled some authors. The base of the cells is usually rather smooth,
though in cases where vessels come into close contact with the
follicle the straight basal line becomes somewhat interrupted
through the influence of the surrounding structures. In Brevoortia
the epithelial cells are nearly all drawn out as if they possess pro-
jections. Those of one follicle approach very closely those of
others and it seems almost as if a connection between the follicles
were established (pl. IV, fig. 1,2). Other somewhat broader cells
possess pedicel-like bases which are sometimes branched, giving
the impression that the cells are sending out pseudopodia. The
processes disappear in the interfollicular tissue in close contact

THYREOID GLAND OF THE TELEOSTS 731

with the blood and lymph capillaries. The process cells are limited
in number and lie close together. Their cell body is swollen with
foamy cytoplasm containing several deeply stained highly refrac-
tive granules. Perhaps these cells are in a state of degeneration,
probably colloidization, although their plasma does not show any
acidophilia (pl. IV, fig. 5, E). Peremeschko observes somewhat
comparable features in the thyreoid glands of birds and mammals,
especially in that of the rabbit. Some of the epithelial cells possess
at their basal end from one to ten small projections, and thus
resemble the tassel cells Pfliiger has described in salivary glands,
except that in the thyreoids the processes are shorter. In some
cases Peremeschko found such cells in fresh material and could
isolate these follicles, which appear to be surrounded by a fringe.
Pfliiger regarded the cell processes as nervous, but Peremeschko
correctly believes them to come from the cytoplasm of the epi-
thelial cells.

The function of the follicles can be much more easily studied in
other groups of vertebrates than the teleosts. In dissecting out
the follicles, as far as they are macroscopically visible, and fixing
them, it is almost impossible to avoid destroying the finer struc-
tures. Hemorrhages are almost unavoidable in cutting open the
gill region. On the other hand, in fixing the entire floor of the
pharynx the fixation fluid does not penetrate sufficiently fast to
preserve the finest details, and the general structures are unfavor-
ably influenced by the decalcification process. Microchemically,
therefore, little can be done and I limit myself to what could be
determined from studies of general structures.

Hiirthle’s colloid cells were seldom seen in the thousands of
follicles observed. Whether they are not generally formed, or
whether they appear and are emptied in so short a time as to be
rarely preserved I am unable to say. In Clupea (pl. V, fig. 20,
Coz.) they were limited to four or five neighboring follicles and
in these all of the epithelial cells were so swollen that insome cases
they met in the center of the follicle, obliterating the lumen. The
nuclei were compact and deeply staining and occurred directly
under the free surface of the cell. The cytoplasm was homogeneous,
highly eosinophile, and sharply distinguished from that of other

732 J. F. GUDERNATSCH

epithelial cells, and thus the colloid forming zone was well defined.
This agrees with Hirthle’s account, which states that the colloid
cells always appear at the same time in a large portion of the wall
of a follicle or in several neighboring follicles. The size of the
follicles has nothing to do with their appearance. In one case,
Siphostoma, the epithelium of all follicles consisted of cuboidal
swollen cells, the nuclei of which were near the cell center or
towards the lumen and the cytoplasm was highly acidophile,
(pl. LV; fig8):

The normal contents of the follicles is the colloid. Itis found
in all thyreoids and usually all the follicles contain it; only in a
few cases were the majority of them empty.

In spite of the various ideas expressed in the literature regard-
ing the surface irregularities of the colloid there is little doubt
that they are caused by shrinkage in fixation. In the majority
of follicles the surface of the colloid was perfectly smooth, in
some a little retracted from the epithelium, but in others com-
pletely filling the lumen. In some follicles the colloid showed
surface indentations. These differences can scarcely be con-
nected with the age of the organ, as they were observed in differ-
ent stages. In all the young trout, however, the colloid filled
the follicles completely. One possibility is that the content of
the follicles does not always possess the same chemical compo-
sition, and is influenced by the same fixation fluid in different
ways.

The view has been held that the true secretion of the cells is
hyaline and that it appears in the form of small droplets which
are set free on the surface of the cells. This process is thought
by some to be responsible for the irregular surface of the colloid.
Two kinds of surface irregularities must be distinguished, first,
the large ones which do not correspond with depressions of the
epithelial cells. These are without doubt due to the fixation.
The connecting threads of colloid between: the central portion
and the epithelium seem to run between the cells and to take
hold there. Tangential sections through the follicle wall, cutting
the epithclium just under the free surface, show that the cells
do not always lie closcly placed in their upper portions and a

THYREOID GLAND OF THE TELEOSTS 733

network of colloidal threads is shown between them. In higher
vertebrates these large surface irregularities in the colloid seem
more common than in fish. The second, smaller irregularities
might resemble secreted droplets. They give to the surface of
the colloid, especially from a top view, the appearance of being
beset with oil drops. In some places there are merely slight depres-
sions in the free margin, some distance apart, while in others
the whole surface is corrugated, but these irregularities do not
appear in all of the follicles. Whether they are really physio-
logical products of the cells is not determined. The irregulari-
ties may be more easily explained on the theory that where the
free ends of the cells do not come in close contact, the colloid
which fills the follicles is pressed into the intercellular spaces
and surrounds the top of the cells like a cap. In shrinkage from
fixation the caps would be pulled from the cells, leaving on the
surface of the colloid the impressions. Anderson regards these
‘droplets’ as well as the numerous vacuoles, which he finds within
the colloid even of living glands, as ‘‘cavities lined with a hyaline
membrane and containing the ‘chromophobe’ secretion, a part
of the secretory activity of the gland.”’ Langendorff and others
more correctly regard them as artefacts, having no physiolog-
ical significance. Vacuoles within the colloidal substance are
seldom seen, (pl. V, fig. 18, V).

The colloidal material seems to become denser with age, as
far as this can be determined by its staining capacity. In young
trout it is rather pink so that it can scarcely be distinguished from
the blood serum in the vessels. Both structures show the same
microscopic appearance. In older trout, however, the colloid
stains very deeply with acid dyes. ‘These observations agree
with those of Schmid on dogs of different ages. Anderson, Boé-
chat, Peremeschko and others also state that the number of
follicles containing a slightly staining finely granular colloid
diminishes with age, being small in old individuals. I failed to
find some follicles distinguished by a greater affinity for the
stain than others, as was claimed by Hiirthle, but did find that
sometimes within the same follicle the colloid stained differently
in different places.

734 J. F. GUDERNATSCH

The structure of the colloid varies with the species and is the
same through all the follicles. A perfectly homogeneous colloid
exists in cases, in others it is granular, and finally in some fish it
is of a lumpy consistency. The consistency of the colloid also
varies with age, in old animals being rather cloudy in appearance
and evidently very brittle after fixation. Occasionally the outer
portion of the colloidal mass stains a trifle lighter which is the
only indication of a concentric structure. This alone, however
does not argue for the view that the colloid is a by-product of
the active thyreoid, which collects and remains in the follicle.
Langendorff first presented such an idea which of course called
forth great opposition.

Blood corpuscles are occasionally found in the Teleost thyre-
oid and sometimes completely fill the lumen of the follicles or
may be scattered or bunched together. Blood is also occasion-
ally found in the human follicles. Baber was no doubt mistaken
when he spoke of a real flow of blood into the follicles, as such
does not occur. How the corpuscles enter the follicles is not
known, though it is probable that somewhere, by pressure or
tension, the delicate wall of a capillary lying next to the epithe-
lium is ruptured and the corpuscles find their way into the fol-
licular lumen through an injured wall. Hiirthle believes the
‘melting’ of the epithelium responsible, when it occurs at a place
where capillaries lie deeply imbedded. It is evident that when-
ever blood corpuscles do enter the lumen they are destroyed,
and they may be seen in all stages of disintegration until finally
pyknotic shadows of nuclei alone exist with no indications of
their cell bodies. The scattered corpuscles lie within the colloid,
which must therefore, be rather liquid. The content of the
follicles has a haemolytic property without being itself of
haematogenic (Baber) origin.

Cells from the follicular epithelium also form a part of the
follicle content. These are pushed off either singly or several
together into the lumen and there destroyed; they also lie within
the colloid. Two kinds of cells are distinguished, those with a
small body and dense cytoplasm, resembling somewhat epithelial
cells of the ordinary type and those with a swollen body, and

THYREOID GLAND OF THE TELEOSTS Tao

clear cytoplasm (pl. V, fig. 13, Coz), which may -resemble
Hiirthle’scolloid cells. The twotypes are probably different stages
in the same process. At times the cell body is broken up into
pieces before being transformed into colloid. The nucleus’ is
always destroyed last.

A part of the colloid is therefore formed by degenerating
epithelial cells which are either destroyed in their primary posi-
tion or after being pushed into the lumen. Anderson believes
that this is invariably the fate of the cells after several periods
of secretion. Hiirthle, also, noticed this ‘melting’ of the epithe-
lium and was able to trace the complete disintegration of the
cytoplasm, though the fate of the nuclei remained doubtful.
They, too, are unquestionably destroyed within the colloid,
and as a matter of fact I could observe cell nuclei, such as those
of the red blood corpuscles, in all stages of disintegration, (pl.
V, fig. 18, N). lL. Miiller regards the formation of colloid
material from disintegrated cells as of slight importance. Hiirthle
remarks that in follicles of mammals with a flattened epithelium,
which he considers the final secreting stage, cell remnants or
defects in the wall can rarely be found. This is equally true in
Teleosts.

I have never seen the signs of degeneration described by Maurer
in old carp. He records a swelling of the epithelial cells which
breaks down the follicles, permitting lymphatic elements to enter
and form lymph nodules, similar to processes in Anura. Perhaps
my specimens were not old enough to show these phenomena.

Pigment was not observed within the follicles, though outside
of them brown pigment is often found in the supporting tissue.
This is probably of haematogenic origin. Baber found brown
pigment granules within the colloid in the thyreoid gland of the
conger eel. I also fail to find crystals in the follicles or around
them as has been reported by some investigators. They are
undoubtedly postmortem products.

In the conger eel Baber observed a reticulum between the
epithelial cells, in which they were partially imbedded. He
states that this reticulum is formed by coagulated intercellular
substance and has nodal thickenings. At the thickened places

736 J. F. GUDERNATSCH

the ‘club shaped’ cells described above are located and may be
clearly distinguished from the ordinary epithelial cells. I did
not find such a reticulum, and it is possible that the filling of
the intercellular spaces with colloid substance as before men-
tioned, may have been what Baber observed. He states that
the reticulum (intercellular substance) stained with hematoxylin,
which makes it very different from the highly eosinophile net-
work observed by me. Baber’s technique, however, seems to
have failed to produce the proper differentiation, since he actu-
ally succeeds in staining parts of the colloid with nuclear dyes.

The disputed membrana propria. was not observed. W. Miil-
ler, K6lliker and others claim to have seen it everywhere while
Schmid and others definitely deny its existence. The connective
tissue approaches the follicles and surrounds them but this loose
connective tissue sheath, which is by no means always present,
could scarcely be called a propria.

The blood supply of the thyreoid gland is abundant and varies
somewhat with the species. Baber is the only observer who has
studied the conditions in the Teleost thyreoid by aid of the injec-
tion method, and unfortunately he used only one specimen of
the conger eel.

The capillaries often approach the follicles so closely as to
seem imbedded between the epithelial cells. This is best shown
when both follicles and vessels are cut in cross section, (pl. IV,
fig. 6). Hurthle describes a similar condition in the thyreoid
glands of young dogs and pictures them in plate II, fig. 6. The
epithelial cells often partly surround the capillaries by means
of processes, thus forming deep impressions. Baber speaks of
small intercellular projections from the capillaries which seem
to serve in retarding the circulation of the blood.

There is usually a network of capillaries around each follicle,
four or five often being seen in cross section just outside the
epithelium, (pl. IV, fig. 5,a). In longitudinal section, the cap-
illaries at times surround almost the entire periphery of a fol-
licle. Such specimens illustrate how closely epithelium and
endothelium are neighbors without a separating basement mem-
brane, (pl. IV, fig. 5, E, Ca).

THYREOID GLAND OF THE TELEOSTS tat

Where the follicles are densely packed, numerous spaces and
channels run between them. The smallest of these seem to have
no endothelial wall, so that the lymph flows directly against the
epithelium of the follicles. In other cases the lymph vessel is
indicated by two parallel endothelial lines running between the
follicles. This does not agree with L. Miiller’s view that the
blood capillaries are in close contact with the epithelium while
the lymph vessels are separated from the follicles by blood ves-
sels or connective tissue. The follicles are sometimes, as de-
scribed in the anatomical part, situated directly on the big lymph
spaces around the ventral aorta as the text figures 4 to 7 show.
(See also pl. IV, figs. 2, 3.)

In the conger eel and skate Baber was unable to detect the
lymph vessels. Since he injected the venous system which is
connected with the lymph vessels he thus regarded the lymph
capillaries as veins. Ferguson has been more successful in dis-
tinguishing between these two sets of vessels in the dogfish.

In some instances, less often, however, than it occurs in higher
vertebrates, a substance was found in the lymph vessles, which
had apparently the same structure as the contents of the follicles.
The lymph spaces were filled with this substance in one instance
and showed many smaller channels running together into the
larger ones (pl. V, fig. 18, LZ). According to Anderson the colloid
in the lymph vessels undergoes a change, becoming diluted and
finely granular and is difficult to distinguish from blood serum.

The way in which the colloidal material leaves the follicle
is not made clear by my study. Attention may be called to the
varying views of different authors, especially those of Biondi
and Anderson, given in their description of the follicular epi-
thelium. It must be mentioned also that Hirthle believes in
temporary intercellular channels which form between the cells
for the passing of the colloid. I saw in a very few cases a colloidal
pseudopodium, as it were, push through the epithelium.

I am also unable to state from the thyreoid gland in the Tele-
osts whether the veins contain colloidal substances and carry
them into the circulation.

738 J. F. GUDERNATSCH
RESUME

The anatomy of the thyreoid gland of the Teleosts is decid-
edly different from that of most other vertebrates. It is not
an anatomical unit. The term ‘thyreoid gland,’ therefore, is
scarcely appropriate. Physiologically isopotent units are dis-
tributed over a wide area. Physical influences must be made
responsible for this distribution, which is due to mechanical
conditions of pressure and pull.

If the thyreoid gland of the Teleosts really have its prototype
in the endostyle of the Tunicates, its phylogeny is somewhat as
follows. We have at first a uniform organ with a given function,
later a change of structure and function takes place, and the
organ loses its unity (Myxinoids and Teleosts). In higher forms
the new function is maintained but the organ retains its original
uniformity and integrity.

The development of the organ from its anlage to the mature
state seems to be simpler in Teleosts than in higher vertebrates.

The histology of the glandular elements of the thyreoid in the
Teleosts is but little simpler than in higher vertebrates. It shows
many parallels to the different features observed by numerous
authors in other thyreoid glands.

The function of the thyreoid, concluding from its micro-
scopical appearance, must be closely the same in Teleosts as it
is in other vertebrates.

THYREOID GLAND OF THE TELEOSTS 739

SPECIAL PART

The species examined were:

ORDER

Apodes
Isospondyli

Hemibranchii
Lophobranchii
Haplomi

Mecho ald

Acanthopteri

FAMILY

Anguillidae
Clupeidae

Salmonidae

Argentinidae
Gasterosteidae
Syngnathidae
Poecilidae

Atherindae
Mugilidae
Scombridae
Pomatomidae
Serranidae
Sparidae
Sciaenidae

Labridae

Tetraodontidae

Triglidae
Batrachoididae
Blenniidae
Pleuronectidae

SPECIES Z

Anguilla chrysypa.
Clupea harengus.
Brevoortia tyrannus.
Oncorhynchus kisutch.
Salmo mykiss.
Salmo irideus.
Cristivomer namaycush.
Salvelinus fontinalis.
Osmerus mordax.
Apeltes quadracus.
Siphostoma fuscum
Fundulus heteroclitus.
Fundulus majalis.
Fundulus diaphanus.
Menidia notata.
Mugil cephalus.
Sarda sarda.
Pomatomus saltatrix
Morone americana.
Stenotomus chrysops.
Cynoscion regalis.
Micropogon undulatus.
Tautogolabrus adspersus.
Tautoga onitis.
Spheroides maculatus.
Prionotus carolinus.
Opsanus tau.
Muraenoides gunellus.
Pseudopleuronectes
americanus.

ANGUILLA CHRYSYPA RAFIN

The thyreoid gland in young eels, 30 cm. long, has a trans-
verse and not a dorso-ventral extension as one might expect in
a species with a narrow floor of the pharynx. It begins far for-
ward in the arterial bifurcation lying close under the basihyale
and extends back to the second gill arteries (plate I, fig. 11.) Close
behind the anterior end of the gland the transverse distribution
of follicles becomes rather wide, (fig. 1, A), extending over the

740 J. F. GUDERNATSCH

Fig. 1. Sections through the thyreoid gland of Anguilla. A, anterior to the aor-
tic bifurcation; B, between the first and second branchial arteries. Thyreoid
follicles in all figures shown in solid black. Transverse muscles lined. Long-mus-
cles in polygons. Skeletal parts stippled. Arteries heavy line. Veins light line.
Lymph sinus broken line. A, ventral aorta. Ay, Ar, Ay, branchial arteries.

entire space between the first gill arches, about 2.6 mm. The
layer of follicles is very thin so that the dorso-ventral extension
is slight. Near the union of the two first gill arteries the follicles
are somewhat more dispersed, and reach out dorsally along the
sides of the basibranchiale. Some follicles actually lie dorsal to
the skeletal parts. The thyreoid is in contact with the first gill
arteries for a short distance, and here it reaches its maximal
extension. Further back it is limited to the neighborhood of the
ventral aorta.

Behind the aortic bifurcation the follicles lie closely above
and to the sides of the aortic stem and extend along it to the
second gill arteries. A string of follicles lies separated between
the first and second arterial branches. Baber states that in the
conger eel the gland is in the first bifurcation and forms a reddish
flattened body. This would correspond to the region of maxi-
mal dispersion of thyreoid follicles in the species here mentioned.

The follicles exhibit a variety of shapes, elliptical ones being
in the majority. They are rather small, 100u representing the
average diameter of the circular follicles. A few very large fol-
licles are present; these ‘giant’ follicles as they might be called,
are of elliptical shape measuring 600u in the long and 200y in the

THYREOID GLAND OF THE TELEOSTS 741

short axis. Baber observed in the conger eel follicles of very
large size.

Some follicles send out branches which widen near their end
to form secondary cavities, (pl. V, fig. 16). In the series-from
which one section is figured, (pl. V, fig. 15), may be found a
large follicle sending out a branch, and further along two fol-
licles (F. f.) connected by a tube (D) of high cylindrical epithe-
lium. The tube represents the branch of the former section and
in another section both follicles are entirely separated. Going
further in the series the small follicle increases in size while the
large one sends out a second branch. Thus around a larger
follicle as a center may be grouped several smaller ones connect-
ing with the original follicle by ‘ducts’ as it were. These ducts
might be compared with the intercalary portions of other glands.
Baber likewise observed branching follicles in the conger eel.
Baber claims that new follicles arise from groups of cells some-
what rounded in form and situated in the epithelial wall of the
larger ones. I was unable to observe such processes. Lymphatics
are present in the thyreoid gland of the eel, although Baber denies
their existence.

Baber records the follicular epithelial cells as highly columnar
in form. I find cuboidal epithelial cells measuring from 10 to
15y high.

Fig. 2. Sections through the thyreoid gland of Clupea. A, in the aortic bifur-
cation; B, between the first and second branchial arteries.

JOURNAL OF MORPHOLOGY, VOL. 21, No. 4.

742 J. F. GUDERNATSCH
CLUPEA HARENGUS L.

In the herring (specimens 30 cm. long) the thyreoid gland is
well developed (pl. I, fig. 2). The triangular region formed
between the floor of the pharynx, bases of the first gill arches and
a ventrally lying cartilage is entirely filled with follicles. The
distance between the floor of the pharynx and the ventral mus-
culature is considerable, while the cartilages of the basibranchiale
are only slightly developed; there is thus sufficient space for a
dorso-ventral distribution of the thyreoid follicles (fig. 2, A, B).
In certain places the first gill arteries are completely surrounded
by follicles; this is also true of the ventral aorta behind the anter-
ior bifurcation (fig. 2, B). Back of the second branchial arteries
the extension of the gland diminishes, and only small follicles
make a complete ring around the aorta, from which rays of fol-
licles go out towards the cartilages and muscles.

The average size of the follicles is about 200u in diameter;
very large ones are not seen. The follicular epithelium is in gen-
eral rather high and varies between narrow cylindrical cells to
broad cubical ones. The cells are not very densely arranged.
In some regions are found a few neighboring follicles with high
epithelial cells which almost obliterate the central follicular
space (pl. V, fig. 20, Coz). Other follicles have lower cells and
all stages exist between these and the normal ones. This suggests
a zone of Hirthle’s colloid-forming cells. The cytoplasm is highly
eosinophile and the nuclei are located near the inner surface of
the cells. In the intermediate stages, where there is a lumen in
the center of the follicle we find in it colloidal material and red
blood corpuscles.

BREVOORTIA TYRANNUS LATROBE

In this species (length 40 em.) there are very interesting con-
ditions in the extension of the thyreoid gland, due to the enor-
mous elongation of the gill region. The distance from the heart
to the anterior aortic bifurcation measures about 5 em. and with
this stretching of the ventral aorta the thyreoid becomes extended
over a long region. The front.end of the gland lies well beyond

THYREOID GLAND OF THE TELEOSTS 743

Fig. 3. Sections through the thyreoid gland of Brevoortia, all anterior to the
aortic bifurcation.

the aortic bifurcation and the posterior end is at the second
branchial arch, (pl. I, fig. 3).

The floor of the pharynx is very narrow, thus there is no chance
for a lateral extension and the thyreoid follicles become dis-
persed only in a dorso-ventral direction. This extension is
sometimes 4 mm. high (fig. 3).

744 J. F. GUDERNATSCH

The parts of the basibranchiale and hypobranchiale are not
compactly developed, the floor of the pharynx being supported
by a scaffold of osseous lamellae, between which a wide-meshed
fatty tissue appears. There is also an osseous tube open at both
ends (representing perhaps a sub-copula) and enclosing the most
anterior portion of the ventral aorta (fig. 3, A). In this tube the
thyreoid gland extends from the branchial vessels towards the
tip of the tongue. In transverse section the gland appears to be
lying within a bony ring which completely separates it from the
parts outside. At certain places, however, there are openings in
this capsule through which the follicles escape into the outside
tissue. Within the capsule are found osseous lamellae dividing
it into several compartments, and thus three or more bunches
of thyreoid tissue may be seen separated by bone.

At the anterior end the follicles lie outside the osseous cap-
sule and are scattered far apart. They are always located in
the neighborhood of either blood or lymph vessels and probably
follow the vessels as paths of dispersion. The follicles are not
always, however, in direct contact with blood vessels.

The osseous capsule lies above the ventral aorta, and we find
thyreoid tissue only above the vessel. The first gill arteries
for a short distance are completely surrounded by very small
follicles. From the aortic bifurcation the follicles extend far
forward into the capsule although there are no large vessels,
thus there seems to be a tendency towards a forward migration.
This is really the only available space into which the thyreoid
can expand, unless it enter the ventral musculature.

The histology of the gland is somewhat different from that in
other fish. The follicular epithelial cells are drawn out into long
processes which come into contact with those arising from the
cells of near-by follicles (pl. IV, fig. 1). This suggests that the
cells of one follicle might communicate through these processes
with those of the adjacent follicles. The only explanation for
this phenomenon is as follows: originally the follicles lie close
together, with their epithelial cells touching, and when the space
between the skeletal parts becomes wider the meshes of the fatty
tissue, in which the follicles are suspended, are pulled somewhat

THYREOID GLAND OF THE TELEOSTS 745

apart, carrying the follicles with them. The cells, which were
in contact with others or with blood and lymph vessels may have
held fast to them, becoming drawn out into long processes.
They thus form a network between the follicles. These bridges
often surround the capillaries.

There ace only a few follicles which have a regular epithelium
with a smooth outline. Outside the bony ring, described above,
the follicles have the usual epithelium with a smooth surface.

In places the epithelium was found to be disintegrating. The
association of the cells seemed rather loose, their surfaces were
also drawn out into long processes like pseudopodia which some-
times divided into two and disappeared in the interfollicular
tissue (pl. IV, fig. 5, #). These cells did not show any distinc-
tion between nucleus and cytoplasm, and their contents was of
a foamy nature and showed two or three compact deeply staining
granules. ‘They were probably cells which having completed
their secretion period were disintegrating.

SALMO IRIDEUS GIBBONS

Specumens 4 cm. long, one month old. Inthe young rainbow-
trout the thyreoid gland begins in the aortic bifurcation and
extends almost to the third gill arteries, (pl. II, fig. 22). There
is little space for a lateral extension, as the cartilages of the basi-
and hypohyalia form a rather narrow arch, and limit the gland to
the space immediately above and below the aorta. At the aortic
bifurcation the copula comes close to the vessel, so that the fol-
licles are pressed away from the median line, and lie close to the
sides of the cartilage. Later the skeletal parts move back, the
space between them becoming somewhat clearer. Half-way
between the first and second gill branches the thyreoid gland
also extends below the aorta, and a large number of the follicles
lie near the second arterial branches. These ventral follicles
are smaller than the dorsal ones (fig. 4, B). Towards the pos-
terior limit the follicles become smaller and fewer and are again
limited to the region above the aorta. Only two or three folli-
cles are seen in a cross section and at the third gill arteries
they have entirely disappeared.

746 J. F. GUDERNATSCH

Fig. 4. Sections through the thyroid gland of Salmo irideus. A and B, from a
specimen one month old. A, just posterior to the aortic bifurcation; B, near the
second branchial arteries. C and D, from a specimen one yearold; C,in the aortic
bifurcation; D, at the second branchial arteries.

The follicles are usually circular but there are also irregular
forms, due to pressure from the surrounding tissues. The diam-
eters of the circular follicles vary between 10 and 80y, the larger
ones being rare. The follicular epithelium is everywhere low,
the cells measuring about 34. The nuclei are all placed with
their broad side towards the lumen, which might be called the
flat cell position to distinguish it from the cylinder cell position
in which the nuclei point towards the lumen.

Almost all the follicles are filled with clear homogeneus colloid,
which has only here and there retracted a little from the wall. Blood
capillaries belonging to the follicles, as observed in other species,

THYREOID GLAND OF THE TELEOSTS 747

are not visible. Around the aorta there are rather large veins
or lymph vessels with extremely thin walls, close to which the
follicles lie (fig. 4, B). There is no tissue (basement membrane)
between the epi- and endothelium, the first being almost as thin
as the latter. The nuclei of these epithelial cells are spindle-
shaped and lie far apart.

Specimen one year old. In this fish the distribution of the thy-
reoid is about the same as in the younger one, (pl. IJ, fig. 23).
The anterior end is pushed further forward in the aortic bifurca-
tion, and the posterior end still lies close to the third gill branches.
The mass of thyreoid tissue is much enlarged. The follicles are
much larger in the bifurcation, and in a section there are more
than three times as many as in a one month old individual. They
are packed more densely and completely fill the space between
the cartilages and arteries. The process of the copula mentioned
above, which comes down to the level of the aorta, here divides
the thyreoid into a right and left half. While in the younger
trout the lateral extension of the follicles was less than the dorso-
ventral, at this age the floor of the pharynx has become broader
through a widening out of the gill arches, and the lateral distri-
bution is more than twice as extensive as the dorso-ventral,
although the follicles still go high up along the cartilages (fig. 4,
C). The follicles also extend some distance along the first
branchial arteries. Here the entire thyreoid lies dorsal to the
blood vessel and is grouped around two or more large lymph
spaces (fig. 4, C). Immediately behind the aortic bifurcation
the lateral and then the dorso-ventral dispersion of the follicles
decrease, so that they lie more densely packed and are fewer in
number.

The hypobranchialia approach closer and closer to the copula
as we pass backward and force the thyreoid to a more ventral
position. Finally the aorta lies almost on the cartilages and the
thyreoid shows only one or two follicles in the section. This
restriction of the thyreoid zone (pl. II, fig. 23) between the first
and second branchial arteries is typical for all salmonids. It may
also occur in some other species but is never so pronounced as in
the trout.

748 J. F. GUDERNATSCH

Near the second branchial arteries the skeletal arch becomes
flattened again, the copula does not reach so far down, and first
the dorso-ventral and later the lateral distribution of the folli-
cles again increases. Comparatively few follicles now appear
below the.aorta (fig. 4, D).’ Behind the second branchial arteries
the follicles decrease in number and size, and completely dis-
appear before the third branchial arteries are reached.

The follicles are circular, oval or irregular in cross section.
The diameters of the circular ones vary between 40 and 200,,
the larger ones are more numerous, especially in the anterior
region. Branched follicles occur, sometimes as many as five
follicles leading into a larger one.

Here also the follicular epithelium is low, almost flat, and the
follicles are completely filled with homogeneous colloidal sub-
stance. Sometimes, however, the colloid contains particles,
probably destroyed blood corpuscles or epithelial cells. The
blood supply is rich, many capillaries lying close to the follicles.
There seems to be a comparatively better circulation here than
in the younger stages.

SALMO MYKISS WALBAUM

Specumen 11 cm. In the black spotted trout the thyreoid
gland shows a great antero-posterior extension. The posterior
limit is about that shown by Maurer in a 20 cm. trout, species
not named, apparently a brook trout. However, the main part
of the gland is situated above the aorta, not below it as Maurer
claimed. The anterior limit of the gland lies well in front of the
aortic bifurcation and the posterior end behind the third branchial
arteries (pl. II, fig. 21). The dorso-ventral distribution is also
more pronounced than in most of the other species, especially as
to the number of follicles below the aorta. The main mass of
the organ lies in the aortic bifurcation (fig. 5, 4). The copula
reaches far down and divides it into two halves. Along this
cartilage the follicles extend dorsally close up to the floor of the
pharynx. Laterally also the extension of the follicles goes as far
as possible.

THYREOID GLAND OF THE TELEOSTS 749

Fig. 5. A to C. Sections through the thyreoid gland of Salmo mykiss. 4A,
in the aortic bifurcation; B, closely anterior to the second; C, at the third bran-
chial arteries. D. Section through the thyreoid gland of Cristivomer at the
second branchial arteries.

The follicles do not lie directly on the first branchial arter-
ies, but are grouped around large veins or lymph sinuses, the
dorsal follicles being much larger than the ventral ones. Behind
the first arterial branches the dispersion of follicles is very much
reduced. They are forced away from the dorsal region by the

750 J. F. GUDERNATSCH

development of the basi- and hypobranchialia and occur only
laterally and ventrally of the aorta. Further back even the lat-
eral follicles disappear and only a few small ventral ones are
grouped around a small vessel. Space again becomes available
towards the second branchial arteries, since the skeletal parts
retract more and more, and the follicles reappear in their former
locations. The lateral extension however is not as great as in
the region of the first arteries, since longitudinal muscle bundles
prevent it (fig. 5, B). Dorsally the follicles again reach up to
the pharyngeal floor. Close to the second branchial arteries
the dorsal extension again diminishes and almost disappears
when the second gill arteries are reached. Here the follicles lie far
below the aorta, as they are forced away from the vessel by a
longitudinal muscle. From this place backward a few follicles
again appear above the aorta; they are small and scarce, five or
six in a section, and widely scattered. Behind the third arteries
the ventral aorta lies buried far beneath a muscle, between which
and the skeletal parts a portion of the thyreoid lies. Another
portion lies below the aorta between the third and fourth aortic
branches, and here once more the amount of thyreoid tissue is
slightly increased. A small mass of follicles disconnected from
the main mass appears behind this place, lying below the aorta.

Cross sections through the follicles are usually circular, some
are irregular. Their size decreases from the anterior towards
the posterior end of the thyreoid region. The diameters vary
between 10 and 60u in asingle section. The epithelium is rather
flat, though some follicles have a cubical epithelium 3 to 5u
high. The nuclei of the flat cells show a peculiar feature; in all
other cases they are either round or oval, but here with a few
exceptions they are bent, taking forms ranging from wide arches
to perfect horse-shoe shapes and are from 8 to 10u long. In all
probability they are degenerating, since they do not stain as
deeply with nuclear dyes as do the round nuclei. Many of the
follicles do not contain colloid.

The blood supply of the thyreoid zone is rich but there are no
capillaries to the follicles proper, although there are smaller
blood vessels in the region. Large veins and lymph vessels lie
around the aorta and the follicles lie close to their walls (fig. 5, B).

THYREOID GLAND OF THE TELEOSTS 751

CRISTIVOMER NAMAYCUSH WALBAUM

Length of specimen, 12 cm. The outlines of the thyreoid region
in the great-lake trout are about the same as in the former spe-
cies, but the ventral and posterior extensions are more limited.
The anterior end lies in front of the aortic bifurcation, the pos-
terior end at the third branchial arteries, (pl. II, fig. 24). The
conditions from the aortic bifurcation to the second branches
are the same as described in the species above but at the second
arteries the accumulation of thyreoid material is rather large.
Here also are found the largest follicles. The lateral extension is
wider than at the first branches. The aorta is surrounded by
follicles (fig. 5, D) but they do not lie very close to its wall. Pos-
teriorly the extension decreases, three to four follicles being seen
in a section above and below the aorta. The ventral follicles
soon disappear and at the third aortic branches the dorsal ones
also run out.

The follicles are a little larger than those of the black trout.
Irregular and circular cross sections of the follicles are seen,
the latter 20 to 100u in diameter. The epithelial cells are gen-
erally cubical, about 6» high. The nuclei are circular, 3y in
diameter, oval or somewhat irregular. The bent nuclei des-
cribed in the black trout are present, but not so numerous. Some
follicles show only regular nuclei, others only irregular, so that
one might imagine these forms associated with different physi-
ological stages. Almost all the follicles contain colloid.

There are many capillaries in the fatty tissue in close contact
with the follicles.. The follicles are not located on large veins
and only a few lie close to the lymph sinuses.

SALVELINUS FONTINALIS L.

Length 4 cm., age 1 month. In this young brook trout the thy-
reoid gland has not developed very far, certainly not so far as
Maurer describes for this stage. The follicles are scarce, the most
anterior lying in the aortic bifurcation. Between the first and
second branchial arteries there are a few follicles in each section,

752 J. F. GUDERNATSCH

situated above the aorta; near the second a few appear below the
aorta (fig. 6, A).

Length 25 to 30 cm. In the brook trout a condition of remark-
ably wide distribution of thyreoid material is seen. The region
of the thyreoid in this species is comparatively larger than in
any other fish. The anterior end of the gland is far in front of
the aortic bifurcation and small follicles extend to the floor of
the pharynx (fig. 6, B).

The first branchial arches are completely unreal by thy-
reoid follicles. In the aortic bifurcation the follicles are very
numerous, densely packed and occupy a rather large field. They
reach up to the dorsal edge of the copula and laterally to the gill
bases. On both sides of the aorta they are scattered between
the fibres of longitudinal muscles (fig. 6, C). The follicles force
their way through’the muscle tissue along blood vessels and con-
nective tissue fibres. Below the aorta their arrangement is less
dense. Close behind the aortic bifurcation the amount of thy-
reoid tissue is reduced in the typical way, the copula extending
down to the aorta. By this arrangement three, more or less
separated, thyreoid masses are formed, two dorsally to the right
and left of the copula and one below the aorta. The ventral part
decreases, then the dorsal masses, the arrangement of the fol-
licles becoming looser. Although the dorsal space becomes more
open the follicles still decrease in size and are scattered far apart,
indicating that this is a zone between two accumulations of thy-
reoid tissue, those around the first and second aortic branches.
Two centers of growth may easily be determined.

Just before reaching the second branchial arteries the lateral
extension becomes very great (fig. 6, D). The follicles migrate
into the first gill arches along the branchial arteries and occur
at the base of and extend into the second gill arches. This wide
distribution of thyreoid elements is certainly the most remark-
able feature of the organ in the Teleosts. Follicles not only le
at the base of the gills, but are distributed along the laminae at
the base of the villi.

At the second branchial arteries the thyreoid gland, as men-
tioned above, once more shows an extensive development. Above

THYREOID GLAND OF THE TELEOSTS tao

Fig. 6. A. Section through the thyreoid gland of a one month old specimen of
Salvelinus fontinalis, between the first and second branchial arteries.

Bto D. Sections through the thyreoid gland of an adult specimen. Band C,
in the aortic bifurcation; D, near the second branchial arteries.

the aorta a dense arrangement of follicles is seen and below the
number increases. Laterally the follicles extend along the arter-
ies. Behind this place the aortic stem is entirely surrounded by
thyreoid tissue which completely fills the space between bones
and muscles. Small follicles are imbedded in the adventitia of
the aorta. There is a dissolution of the dense arrangement in
the peripheral zones, especially ventrally. Close behind the
second branchial arteries the mass of thyreoid tissue decreases
very suddenly and only a thin ring of follicles surrounds the aorta.
Towards the third branchial arteries the aorta becomes buried
between the ventral muscles, and the ventral follicles disappear
sooner than the dorsal ones which continue and surround the
third branchial arches for a short distance. Behind the third

754 J. F. GUDERNATSCH

branchial arteries a small accumulation of thyreoid tissue once
more appears.

A second series shows conditions similar to those above de-
scribed. The follicles in the anterior region are less densely
arranged. The basibranchiale comes very close to the aorta and
separates to some extent two portions of thyreoid tissue along
the aortic stem. The follicular mass is little reduced behind the
aortic bifurcation but a little in front of the second branchial
arteries the typical restriction is found. At this place the first
ventral follicles appear. When the second aortic branches are
reached the lateral extension of follicles becomes very wide.
The mass of thyreoid tissue is here much increased, and the
ventral portion is well developed but not so far as in the trout
described above. The thyreoid stops close behind the second
branchial arteries.

In a third series the separation of follicles in the anterior por-
tions is still greater than described in either the first or second.
The dorsal limit reaches to the upper edge of the basihyale,
where there is an accumulation of follicles on both sides. The
first branchial arteries are for a long distance completely sur-
rounded by follicles, but the number of follicles decreases visibly
towards their union; thus in this case there is an accumulation of
follicles in front of the first aortic bifurcation. The ventral fol-
licles appear at the first branchial arteries and disappear before
reaching the second. It seems that here the entire thyreoid mass
is pushed much farther towards the head than in the other trout
described. Between the first and second branchial arteries the
conditions are similar to those in the other specimens, the distri-
bution of the follicles being restricted. There is no pronounced
increase of thyreoid tissue or lateral distribution at the second
branchial arteries and the posterior limit of thyreoid follicles is
in front of the third branchial arteries.

High epithelial cells were predominant in the follicles of all
the thyreoids. The cubical cells measure 9 to 10u broad and
12u high, and the narrow cylindrical cells are 2 to 3u broad
and 20u high. The nuclei are usually large and round, except
in the very high cells where they are compressed. In a few places

THYREOID GLAND OF THE TELEOSTS ta

not all the nuclei of a follicle show the same structure or the same
reaction towards the stain and thus may be in different physi-
ological stages. In addition to normal, large nuclei with distinct
nucleoli and granular structure we find compact deeply staining
nuclei which sometimes contain a vesicle. There are also small
pyknotic nuclei in small (degenerating) cells. Often such com-
pact nuclei with a halo of colloid are found within the lumen and
it seems then that the epithelial cells have emptied their entire
content. These masses can be easily distinguished in the colloid
even after their outlines become indistinct as they have a differ-
ent refractive index. Maurer describes somewhat similar struc-
tures in trout and carp. In other cases several neighboring cells
with much swollen bodies have been pushed off from the epi-
thelium and may be seen in the colloidal substance (pl. V, fig.
bey)

The general form of the follicles is globular, though the sur-
rounding fat and muscle tissue influences the outlines to some
degree (pl. V, figs. 10-12).

Smooth muscle fibres are found in the entire thyreoid region;
in one case (the first specimen) only ventral to the aorta. They
run in all directions in the interfollicular tissue. The follicles are
often arranged along them or are surrounded by them. Where
the follicles lie in clusters of five or ten or more, smooth muscle
fibres are found running between them. The muscle fibres with
the follicles, their capillaries and the connective tissue fibres
form a somewhat compact structure.

The blood supply to the secreting epithelium is extremely
rich, several capillaries going to each follicle (pl. V, figs. 10, 12
Cai:

The thyreoid gland in two other species was dissected out as
far as it was visible macroscopically. In this way of course one
does not get the scattered follicles but only the main masses.
Figs. 28 and 29 of plate III from these two dissections as well
as figs. 25 to 27 of plate II, which are from specimens cut in
serial sections, show that the distribution of the thyreoid in the
trout is very variable.

756 J. F. GUDERNATSCH
ONCORHYNCHUS KISUTCH WALBAUM

Specimen 6 months old, 7 cm. long. The thyreoid gland in the
silver salmon extends further back than in most of the trouts,
reaching beyond the fourth branchial arteries (pl. V, fig. 20).
Another, feature in the arrangement is that the follicles lie rather
close together, surrounding the stem of the ventral aorta through-

Fig. 7. Sections through the thyreoid gland of Oncorhynchus. A, just posterior
to the aortic bifurcation; B, at the second branchial arteries; C, posterior to the
third branchial arteries.

out almost its entire length. The amount of thyreoid tissue is
small at the aortic bifurcation and between the first and second
branchial branches (fig. 7, A). At the second gill artery the thy-
reoid tissue is most abundant.

THYREOID GLAND OF THE TELEOSTS 757

In front of the aortic bifurcation the basi- and hypobranchia-
lia reach down and here only a few follicles are found on both
sides of the hypohyalia. Back of the place where the cartilages
have retracted the longitudinal muscle bundles prevent the lat-
eral expansion of the thyreoid (pl. V, fig. 14). At the second
branchial arteries however, the mass of thyreoid tissue is very
much increased, again surrounding the aorta (fig. 7, B). The
ventral extension is pronounced. As a rule in the trouts no fol-
licles lie directly against the aortic wall but here there is a com-
plete ring of them around it. Above this ring lies a large lymph
sinus and between it and the skeletal parts thyreoid tissue is
again found. ‘Towards the third aortic branches the cartilages
again compress the aorta, and here the follicles lie around the
aorta and along the outlines of the cartilages. Further back
the aorta sinks down between the muscles, the ventral follicles
disappear and the dorsal ones do not follow the vessel, but increase
in number and group themselves around a subcopula between
the third branchial branches. This dorsal rather compact group
of follicles extends back behind the fourth branchial arteries.
Below the aorta a small cluster of four or five follicles appears
as is seen in other species of trout (fig. 7, C). At the level of the
fourth aortic branches the copula extends so far ventrally that
the dorsal follicles are pressed between the muscles and again
come down into contact with the aorta. The posterior end of
the thyreoid is in this region of the fourth arch.

The form of the follicles is usually elliptical, though circular
cross sections are also found, ranging from 15 to 100 in diame-
ter. The larger ones are more abundant.

The follicular epithelium is very flat (pl. V, fig. 14), and in
most of the cells are again seen the irregular nuclei described in
some of the above species of trout (pl. IV, fig. 9, V). The major-
ity of follicles are in close relation with large lymph sinuses, epi-
and endothelium being in contact (fig. 7, B, C). There are no
capillaries to the follicles proper.

JOURNAL OF MORPHOLOGY, VOL. 21, NO. 4

~]
on
Go

J. F. GUDERNATSCH
OSMERUS MORDAX MITCHILL

A smelt 20 cm. long. This smelt presents the thyreoid condi-
tions described below. A few follicles appear far in front of the
aortic bifureation (fig. 8, A), and further back more are arranged
around the copula. At the bifurcation every section shows
twelve to fifteen follicles between the stem of the aorta and the

Fig. 8. Sections through the thyreoid gland of Osmerus. A, anterior to the
aortic bifurcation; B, just posterior to it; C, between the first and second; and D,
close to the second branchial arteries.

copula. The follicles vary in size from 40 to 200u. Behind this
the main mass of thyreoid tissue lies above the aorta, a few fol-
licles lie to either side, and ventral to the aorta they are very
searce. Further back the basibranchiale comes nearer and nearer
the aorta, finally reaching it, so that the follicles are forced out
laterally (fig. 8, C). In the region of the hypobranchialia are

THYREOID GLAND OF THE TELEOSTS 759

seen only a few follicles far to the sides of the aorta and skeletal
parts. Behind this the two hypobranchialia have retracted a
little from the copula and very small follicles appear in the crev-
ices between them. As the copula retracts from the aortic stem,
more follicles appear on the dorsal side of the aorta. At the
second branchial arteries the follicles cease (pl. I, fig. 4). The
follicular epithelial cells are low cuboidal with the longer axis
parallel to the base. The colloid appears homogeneous.

SIPHOSTOMA FUSCUM STORER

Specimen 30 cm. long. In the pipe-fish the thyreoid gland con-
sists of entirely isolated follicles, lying above and to the sides of
the aorta (fig. 9). The external form of the fish influences, of
course, the form of its inner organs. The thyreoid gland has not
found room for dorsal, ventral or lateral expansion and there-
fore extends far backwards as a rather narrow streak. The anter-
ior end lies at the aortic bifurcation and the posterior end close
to the bulbus arteriosus (pl. I, fig. 5). Thus we have a condition
in which the organ reaches further towards the tail than usual
and where the thyreoid region tapers towards the head end, while
as a rule the reverse is true. The number of follicles is not very
large, five or six to the section behind the aortic bifurcation. The
number decreases towards the second branchial arteries and still
more so towards the third, where a transverse muscle occupies
the space between the bones and the aorta. At this place there
are only one or two follicles in a section, yet there is a continuous
chain of them. Near the third branchial arteries the aorta goes
down ventrally, the transverse muscle has decreased, and thus
the thyreoid finds more space for development. There are six
or eight follicles in a section and they lie between the third gill
branches which run dorso-laterally. Behind this place the dis-
persion of follicles increases (pl. IV, fig. 7), although the aorta
lies far ventrally, a fact showing that the thyreoid follicles do not
necessarily use the aortic stem as a migration path. On each
side of the median line a muscle runs in an antero-posterior direction
upon and under which the thyreoid follicles lie. The ventral

760 J. F. GUDERNATSCH

group consists only of small follicles which have traveled down-
wards along a vein running between the two halves of the muscle.
Further back the muscle bundles separate and here the greatest
mass of thyreoid tissue is found. The space between the pharynx
and bulbus is well filled by follicles which lie in a chaos of capil-
laries (pl. IV, fig. 7, Ca, F).

Histologically this gland is as different from that in other spe-
cies as itis anatomically. The gland, when fixed, may have been

Fig. 9. Sections through the thyreoid gland of Siphostoma. A, at the second;
and B, posterior to the branchial arteries.

in a peculiar state of function since there are no reasons to assume
that the histological structures observed are permanent. Colloid
was not found in any of the follicles, at least not as a uniformly
compact mass. Certain follicles contained highly acidophile
lumps about the size of epithelial cells. The epithelium, however,
seemed in a state of colloidalization. The cells were high, cuboidal
and swollen, with bulged out bases and surfaces (pl. IV, fig. 8).
The nuclei were centrally located or towards the lumen. Thus
they seemed to be typical colloid forming cells. The nuclei are in
some cases round and massive, usually however they are very
irregular. In some it seemed as though amitosis was taking place.

THYREOID GLAND OF THE TELEOSTS 761

The formation of colloid ordinarily occurs in only a part of the
thyreoid at a time. Here, however, the entire gland seemed to be
in a similar physiological state.

FUNDULUS HETEROCLITUS L.

Specimens 10 cm. in length. ‘The follicles in the region of the
aortic bifurcation are grouped around a vein, most of them lying
to the sides of it and under a transverse muscle. The elliptical
shape of the vein in sections indicates the pressure between this
muscle and the m. sternohyoideus which forces the follicles out
from the median line. The follicles become more numerous to-
wards the aortic bifurcation and they extend part way out along
the first branchial arteries, and more on their ventral than dorsal
side. Between the first and second gill branches follicles are found
under and above the transverse muscles around which they have
traveled. The ventral aorta in this region is completely sur-
rounded by thyreoid tissue, more being found on the sides than
either dorsally or ventrally (fig. 10, B). At the second gill branches
the follicles again spread out laterally. Behind this place only
a few scattered follicles are found (pl. I, fig. 6).

The size of the folliclesvaries extremely. The smallest are found
at the anterior end and the largest in the middle of the thyreoid
region. They are either circular in cross section, oval or with
irregular evaginations.

The epithelial cells are usually cubical, but in very small folli-
cles sometimes columnar, while in large empty follicles the cells
are flat. Narrower cells with spindle shaped nuclei are seen in
places.

The colloid is granular, and in some regions is seen to leave the
follicle. Whether this is due to artificial pressure cannot be stated.
Occasionally two neighboring epithelial cells will flatten out
somewhat as if they were about to form a passage between them.

The blood supply to the thyreoid region is rich. The follicles
are almost completely surrounded by a net of capillaries. These
vessels are so pressed against the follicle that they form grooves
in it (pl. IV, fig. 6). The projections of the epithelium between

762 J. F. GUDERNATSCH

; &

Imm

Fig. 10. Sections through the thyreoid gland of Fundulus. A to C, F. hetero-
clitus. A, in the aortic bifurcation; B, anterior to, C, at the second branchial

arteries. D, F. diaphanus, 1n the aortic bifurcation. E and F, F. majalis, anterior
to the aortic bifurcation.

the capillaries show narrower and longer cells, and some of these
cells entirely lose their communication with the follicular lumen.
The connective tissue sometimes forms an almost complete
sheath around the follicles and their capillaries. Red blood cor-
puscles in all stages of disintegration are found in many follicles

THYREOID GLAND OF THE TELEOSTS 763

(see General Part). How these corpuscles get into the lumen could
not be determined. Erythrocytes are often seen partially im-
bedded in the follicular epithelium as if they would force their
way in between two cells. In other places corpuscles are found
pressed against an epithelial cell which has so flattened out that
only a thin layer of cytoplasm separates the corpuscle from the
lumen.

FUNDULUS DIAPHANUS LE SUEUR

A specimen 9 cm. long. The main mass of the thyreoid is located
a little nearer the tip of the tongue than in F. heteroclitus (pl.
I, fig. 7). The posterior end lies at the second branchial arteries
where the follicles become scarce and scattered. A further differ-
ence from heteroclitus is that the main mass of follicles always lies
above the aortic stem, only a few small ones lying below. The
lateral extension is here also unimportant.

The floor of the pharynx is narrow and the connection between
it and the ventral musculature is only a narrow streak. In
heteroclitus the lateral pharyngeal axis is the longer one, therefore,
the lateral thyreoid extension prevails; while in diaphanus the
dorso-ventral axis is longer, and here the extension of the thyreoid
is mainly in this direction. Ventrally, however, it is prevented by
the narrow isthmus, and follicles are mainly found above the aorta
(fig. 10, D). In this way the distribution of the follicles may be
figured out mechanically in almost every case.

The follicles are of all sizes, though not so large asin heteroclitus.
There are more elliptical or irregular ones and these have a longer
axis. The cuboidal cells of the follicular epithelium are not as
high as in heteroclitus and cylindrical ones are not found. The
colloid is homogeneous and the blood supply is not rich.

FUNDULUS MAJALIS WALBAUM

Length of specimen 9 cm. The follicles spread out laterally much
further than in the other two species (fig. 10, #, F). They extend
for a distance along the first aortic branches. Between the first
and second branches there is only a narrow streak of thyreoid

764 J. F. GUDERNATSCH

tissue, but the main mass of the organ lies at the second gill branches
and here the greatest lateral extension occurs under a transverse
muscle. The vertical extension is small and there are no follicles
below the aorta. Behind the second gill branches is found the
posterior limit of the gland (pl. I, fig. 8).

The follicles are still smaller than in diaphanus and more
uniform in size. The circular type predominates and they are
more numerous than in the other species. The colloid is homo-
geneous and the follicular epithelium similar to that in diaphanus.

MENIDIA NOTATA MITCHILL

Length of specimen 10 cm. The thyreoid mass is rather small
(pl. I, fig. 9). -The follicles are extremely small, 20-25, and are
scattered along the stem of the aorta between the first and second
branchial arteries and out along the second arteries. The lateral
extension is greater than the antero-posterior.

MUGIL CEPHALUS L.

A mullet 15 cm. long. Small follicles are found in the anterior
end of the thyreoid region and are grouped around a vein (fig. 11,
A). At the aortic bifurcation the organ is better developed, but
is hardly in contact with the gill vessels (fig. 11, B). The thyreoid
lies above the aorta, and at the second branchial arteries it comes
into contact with the vessel. Here the gland is well developed
with numerous large follicles. The follicles disappear towards the
third aortic branches (pl. I, fig. 13).

The size of the follicles varies between 30 and 140 u. In section
they are slightly oval. In the follicular wall are found transi-
tions from flat to high epithelium. The height of the cells varies
within the same follicle, showing that it is independent of follicle
size. The height of the cells rather depends upon outside pres-
sure, e.g., a follicle pressed into oval shape by cartilage shows low
epithelium on the longer sides and higher cells on the short sides.

THYREOID GLAND OF THE TELEOSTS 765
SARDA SARDA BLOCH

Length of specimen 50 cm. The thyreoid gland of the Spanish
mackerel shows the most remarkable conditions of all fish thyreoids.

The mass of the organ is enormously large and the dorso-ventral
and cephalo-caudad extensions are unusual. The relation of the
thyreoid gland to other tissue is singular, and could be compared
only with that in Brevoortia. There exists such an intermingling
of thyreoid, bone, cartilage, smooth and striated muscle fibres,
fat and connective tissue that it is impossible sharply to define

Fig. 11. Sections through the thyreoid gland of Mugil. A, anterior to; B, at
the aortic bifurcation; C, near the second branchial arteries.

766 J. F. GUDERNATSCH

the organ. Yet on the other hand, there can hardly be found a
group of follicles detached from the main thyreoid body.

The isthmus is long, as in Brevoortia, and hence the thyreoid
region is much elongated (pl. I, fig. 10), measuring 4 cm. in length.
It is not, however, as narrow asin the menhaden, having a wide
lateral extension. The anterior end is pushed far forward, 2.5 cm.
in front of the aortic bifurcation, so that it also comes to lie in front
of the hyoid arch. The entire development of the organ takes
place more cephalad than usual and the main mass lies in front of
the aortic bifurcation (sharks!), deeply buried in the body of the
tongue, as a consequence of the ventral extension of the copulo-
hyoid (fig. 12, A). It occupies a more ventral position than any
other fish thyreoid. The follicles are located around a large vein
and arerather closely arranged. As the basi- and hypohyalia
retract the follicles creep into the clefts between them and thus
the thyreoid mass assumes the shape of a horse-shoe, the two
arms of which point dorsally (fig. 12, B, C). The smooth muscle
fibres of this region are completely invaded by follicles (pl. IV,
fig. 21), as are also the bones of the gill arch, especially the copula,
in regions where they lose their compactness and break up into
lamellae. The thyreoid takes the form of three masses converging
ventrally, and as we pass back it expands more and more on the
sides, 6 to 7 mm., while the median branch becomes smaller.
About one em. in front of the aortic bifurcation the most extensive
region of the gland is reached. In cross section the mass is rhom-
boidal, the diagonals being about 7 and 4mm. The lateral exten-
sion decreases while the ventro-median mass increases, from which
two branches tend: dorsally along the edge of the copula. Thus
again the sections show a horse-shoe shape, with a broad middle
piece and narrow dorsally converging arms, in which the follicles
are oval with their longer axis parallel to the line of extension. On
reaching the first branchial arteries, which run in this species
towards a ventro-lateral zone and do not come into contact with
the follicles (fig. 12, C) we pass to their union where a few follicles
surround them (fig. 12, D). The central portion of the gland
becomes smaller and lies separate in the aortic bifurcation while

THYREOID GLAND OF THE TELEOSTS 767

Fig. 12. Sections through the thyreoid gland of Sarda. A, near the anterior
end of the gland; B, in the region of greatest extension anterior to the aortic bifur-
cation; C and D, close to the aortic bifurcation; HZ, near the second branchial
arteries, the region of greatest extension.

the lateral parts increase posteriorly. Thus in the sections there
are three portions of thyreoid which become more and more sepa-
rated by the enlargement of the copula (fig. 12, D). Behind the
aortic bifurcation some follicles appear below the aorta; the middle
mass again enlarges and the three parts unite. One branch again
extends into the copula and soon becomes smaller, while the lateral

768 J. F. GUDERNATSCH

portions increase. The posterior end of the gland is found behind
the second gill branches.

The follicles are usually circular or oval in cross sections, though
many are polygonal from pressure. Their size varies between 30
and 3504 medium sizes being most abundant. Giant follicles
reach 800u long by 400z in short diameter. Many follicles are
without colloid, while in others the colloid is much more shrunken
than usual. The colloid is homogeneous. The follicular epithe-
lium is of high cylindrical cells or cubical ones. The cytoplasm is
stained more darkly in the basal portions; in the higher parts it is
sometimes reddish. The blood supply is rich.

Fig. 13. Sections through the thyreoid gland of Pomatomus. A, anteriortothe
aortic bifurcation; B, between the second and third branchial arteries.

POMATOMUS SALTATRIX L.

Young bluefish 30 cm. long. In this species the dispersion of the
thyreoid follicles is prevented in both a lateral and dorso-ventral
direction, since the arch formed by the basibranchiale and hypo-
branchialia is very narrow (fig. 13). The gland is thus a long
narrow streak (pl. I, fig. 11). At the aortic bifurcation there are
only a few follicles, some of which lie close to the first gill arteries,
just in front of their point of union. The thyreoid mass reaches its
maximum extension above the ventral aorta and between the
first and second gill arteries, especially towards the second. But

THYREOID GLAND OF THE TELEOSTS 769

even here there are only ten or fifteen follicles in a cross section.
Some follicles lie close to the base of the second gill arteries and
from this point the gland extends, with from six to ten follicles
in a cross section, to a little behind the third branchial arteries
where it ends. The follicles are generally dorsal to the ventral
aorta (fig. 13, B), only a few being below it.

The form of the follicles is irregular, but approaches the globu-
lar type. Their size ranges from 15-100y in diameter though
some are far above this size (giant follicles). The minute histology
shows no peculiarities. The epithelium is usually cubical, the
cells being 6y high.

MORONE AMERICANA GMELIN

Specimen 35 cm. in length. The thyreoid gland of the white
perch is characterized by the enormous size of nearly all the
follicles as well as by their unusually loose arrangement. Cepha-
lad of the aortic bifurcation there is little room for dispersion
since the copula reaches far down and the skeletal arch is rather
narrow. Behind the bifurcation (fig. 14, B) this arch becomes
wider and from here to the second gill arteries the main mass of
the thyreoid is situated (pl. I, fig. 12). From the second branchial
arch towards the third two narrow lines of follicles run along the
sides of the aorta. The entire length of the thyreoid region meas-
ures 3.5cem. The majority of the follicles lie above the aorta ex-
cept in the anterior region.

The size of the follicles varies from 120 to 600u in diameter,
the very large ones are most abundant especially in the more
anterior region. In cross sections the follicles are almost all circu-
lar. The epithelial cells are low, 3 to 4u high. In these follicles
there are no indentations in the colloid, it either fills out the lumen
completely or is retracted from the epithelium and has a smooth
edge. (The differences in the colloid of different species may be
of some physiological significance.)

770 J. F. GUDERNATSCH

Fig. 14. Section through the thyreoid gland of Morone. A, at theaortic bifurca-
tion; B, between first and second; C, close to the second branchial arteries.

Fig. 15. Sections through the thyreoid gland of Stenotomus. A, in the aortic
bifurcation; B, at the second branchial arteries.

THYREOID GLAND OF THE TELEOSTS cat
STENOTOMUS CHRYSOPS L.

Length of specimen 25 cm. The scup presents the thyreoid gland
as a rather continuous organ, only one group of follicles lying
below the aorta is isolated from the main mass. The largest
expansion of the gland is in (fig. 15, A) and immediately behind
the aortic bifurcation; here it measures 3 mm. in width, and dorso-
ventrally over1 mm. This expansion is followed by a restriction,
the follicles always lying above the aorta. At the second branchial
arches another increase in the thyreoid tissue occurs, and here
a few follicles appear below the aorta (pl. I, fig. 14).

The size of the generally circular follicles varies from 20 to
300u in diameter, a few reaching 400z.

CYNOSCION REGALIS BLOCH

Specimens of 60 cm. in average length. Twelve specimens of
the squeteague were examined and they serve to show a series
of variations in the thyreoid gland within the species. The region
of the gland extends from in front of the aortic bifurcation to the
third branchial arteries. The majority of follicles always le
either dorsal or lateral to the aortic stem and in only two cases
were any follicles found below the aorta. In one case the aortic
stem between the first and second branches was surrounded. The
region of the second aortic branches is commonly filled by the
gland. The tendency to extend from this place anteriorly is
more often expressed than in the opposite direction. The lateral
extension is greater along the branchial arteries than in inter-
mediate regions (pl. III, figs. 31-41).

In some of the specimens there were two (pl. III, figs. 33, 36,
37, 40) or even three and four (pl. III, fig. 34) well developed
isolated portions of the gland lying on different branches of the
gill vessels. Macroscopically they appear to be separated, but on
tracing the entire region in serial sections it is found that follicles
spread out and connect the several masses, although the follicles
are small and scattered so thinly that they were not seen with the
naked eye.

bo

J. F. GUDERNATSCH

~J
ba |

Fig. 16. Sections through the thyreoid gland of Micropogon. A, between the
first and second; B, at the second; C, near the third branchial arteries.

The mass of thyreoid tissue, roughly judging, differed in the
specimens, although they were of about the same size, yet the
fish may have been of different ages.

The shape of the follicles varies from very irregular to circular.
Their size also varies extremely. Those lying nearest the vessels
are huge and irregular, while the small peripheral ones approach

THYREOID GLAND OF THE TELEOSTS 773

a globular shape. This indicates that the shape of the follicles
is a result of the pressure directions. The follicular arrangement
is rather compact in the central portions. (pl. V, fig. 17.)

The epithelial cells vary from low cuboidal to high cylindrical
shapes. The smaller follicles seem to have a little higher epithe-
lium, though it is rather uniform in the same individual and varies
more among the several specimens. It may seem therefore that
the entire gland is in the same physiological stage.

MICROPOGON UNDULATUS L.

A croaker, 30 cm. long. Thethyreoid extends from the first to the
third branchial arteries (pl. II, fig. 15). The dispersion of folli-
cles is largely dorso-ventral, since laterally they are hindered by
the narrowness of the isthmus (fig. 16, A, C). For this reason also
a considerable part of the gland lies below the aorta, yet not so
large a portion as above, though the dorsal follicles are less densely
arranged.

There are only a few follicles in front of the aortic bifurcation,
yet at the bifurcation and behind it lies the mainmass of the gland.
The follicles completely fill the spaces between bones and vessels
(fig. 16, A). Towards the second gill branches the copula extends
further and further down and forces the follicles into a somewhat
lateral position. The ventral mass is larger in this region. At
the second arterial branches there is no special increase in mass,
the number of ventral follicles having decreased (fig. 16, B), the
dorsal ones increasing and soon extending to the epithelium of
the pharyngeal floor. The follicles lie rather loosely arranged, but
have not noticeably increased in size. A small line of follicles
above the aorta extends from here towards the third gill branches,
others are scattered irregularly around the aorta. The aorta has
sunk into the ventral muscle and carries the posterior follicles
with it.

The thyreoid gland of Micropogon is characterized by rather
small follicles of almost uniform size, though in some regions
large ones appear. The diameters range from 10 to 300, but
those of 30 to 50u are most abundant.

JOURNAL OF MORPHOLOGY, VOL. 21, NO. 4

774 J. F. GUDERNATSCH

The epithelium is rather low, even in the smallest follicles.
Branched follicles are numerous. The blood supply is rich, many
capillaries being present around the follicles. There are several
larger veins running through the thyreoid region.

* TAUTOGOLABRUS ADSPERSUS WALBAUM

Length of specumen 25 cm. In the cunner the thyreoid gland
occupies a unique position, almost resembling that in the sharks.
It is pushed far forward in the aortic bifurcation, and touches both
the first branchial arteries laterally (fig. 17, B), but does not
extend far enough back to come into contact with the ventral
aorta (pl. II, fig. 16). The main mass is, as it were, imbedded in
a bony capsule. The follicles are grouped around a median vein
(fig. 17, A). Dorsal to the aortic bifurcation the copula and a
transverse muscle are well developed, so that the thyreoid is forced
forward. The follicles are not numerous, and are all more or less
irregular. Their diameters measure from 15 to 200u. The
follicular epithelium is cuboidal.

TAUTOGA ONITIS L.

Specimen 35 cm. long. In the closely related tautog the thyreoid
gland also occupies a rather cephalad position (pl. II, fig. 17).
It extends back from within the aortic bifurcation almost to the
second branchial arteries. It lies chiefly above and to the sides
of the aorta. The anterior, main part, is imbedded in an osseous
capsule which is square in cross section, and is formed by three
branchial bones above and a ventral supporting bone (fig. 18, A).
At the aortic bifurcation the capsule becomes incomplete and the
follicles are widely dispersed over 6 mm. (fig. 18, B). The folli-
cles follow the dorsal side of the first arterial branches out to the
base of the gills. Behind the first branchial arteries the lateral
extension decreases and the follicular dispersion is in a dorso-ven-
tral direction. The follicles are loosely arranged, and yet globu-
lar ones are rare, most of them being polygonal in outline. The
average size is 150u in diameter, but there are a few giant folli-

THYREOID GLAND OF THE TELEOSTS “ai

Fig. 17. Sections through the thyreoid gland of Tautogolabrus. A and B,
anterior to the aortic bifurcation.

Fig. 18. Sections through the thyreoid gland of Tautoga. A, anterior to; B,
at and C, posterior to the aortic bifurcation.

776 J. F. GUDERNATSCH

cles, 700u long by 400u broad. Branched follicles are numerous.
The epithelial cells are cuboidal in shape.

PRIONOTUS CAROLINUS BLOCH

Ina sea-robin 30 cm. long the thyreoid gland seemed to show
a pathological appearance. The invasion of the surrounding
tissues by thyreoid follicles was extraordinary, but may be abnor-
mal. For this reason it can only be stated that the gland in this
species occupies a posterior position, close to the origin of the
truncus arteriosus.

OPSANUS TAU L.

A toadfish 30 cm. long the gill region in this species is extremely
shortened, and therefore the thyreoid region begins rather far
forwards. Anteriorly the largest follicles lie on both sides of a
process of the copula which extends ventrally (fig.19, A). Towards
the aortic bifurcation the size of the follicles decreases and the
two lateral portions unite in the median line, at the same time the
lateral extension (fig. 19, B) of the follicles increases remarkably
(pl. II, fig. 18). Some follicles appear below the aortic stem.
Between the first and second branchial arches the number of
follicles decreases above the aorta, while ventrally they disappear
entirely. Along the second branchial arteries the follicles again
reach laterally and also again appear ventrally. Behind this
point the aorta sinks more and more and the space around it
becomes freer. Yet there is no special increase of thyroid tissue
in this region, there being only loosely scattered small follicles.
A. few follicles accompany the aorta in its course into the space
between the musculus sternohyoideus. The caudal end of the
thyreoid lies behind the third gill branches.

The arrangement of the follicles is loose, and they are usually
circular in cross sections. Some are flattened between the bony and
muscular surrounding tissues. Their size varies extremely. The
largest ones, 600 in diameter, lie in the anterior end, which
is the reverse of the general rule for other species. In other regions

THYREOID GLAND OF THE TELEOSTS rae

the follicular diameters vary from 50-400u, the: median size
follicles being the most abundant.

The follicular epithelium is always cuboidal. Colloid is present
in almost all the follicles, and is very brittle and homogeneous.
In the larger follicles the colloid stains much lighter than in the

as
CO DS Oy, ow
DIAS NG ZA A0No!
Iprave pipe) avant wien

Fig. 19. Sections through the thyreoid gland of Opsanus. A, anterior to; B,
at the aortic bifurcation; C, at the second branchial arteries.

smaller. Lymphocytes are numerous within the follicles. The
blood supply of the thyreoid region is very poor.

MURAENOIDES GUNELLUS L.

Length of specimen 40 cm. The thyreoid gland in the butterfish
reaches a considerable size (pl. II, fig. 19). The anterior end
lies in front of the aortic bifurcation and consists only of small

778 J. F. GUDERNATSCH

Fig. 20. Sections through the thyreoid gland of Muraenoides. A, anterior to
the aortic bifurcation; B, between the first and second; C, close to the second
branchial arteries.

scattered follicles. In the middle portion of the gland the folli-
cles are numerous and closely arranged. At the aortic bifurcation
they lie around a large vein and completely fill out the space
between the gill arch and muscles. The lateral extension of the
follicles is small as compared with the dorso-ventral, since the
isthmus is narrow (fig. 20, B). A cross section through the thy-
rcoid area measures about 1 mm. Taking 1 mm. as the average
width we would have 10 cubie mm. of thyreoid tissuein this species.

THYREOID GLAND OF THE TELEOSTS 779

Fig. 21. Sections through the thyreoid gland of Pleuronectes. A, in the aortic
bifurcation; B, just posterior to it.

The first branchial arteries are partly surrounded by follicles.
Behind the aortic bifurcation there is an open space for lateral
extension, but not for ventral, since the aorta rests on the muscula-
ture. The caudal end of the gland lies a little behind the second
branchial arteries, and consists again of small scattered follicles
(ae 20, C).

The follicles are of globular or long ovoid shape, some are very
irregular. The circular cross sections vary from 20 to 500u in
diameter. The very large ones lie at the second arterial branches.
Branched follicles with connecting channels between them are
numerous, so that almost all follicles may be traced in sections
as evaginations of others.

The follicular epithelial cells vary from highly cylindrical,
narrow shapes to broad cuboidal. Flattened epithelium is rare.
The cells are extremely numerous and densely arranged. The
nuclei are located near the base of the cells, even inthe higher ones,
and the cytoplasm stains darker about the nucleus. Sometimes
it appears as if there were a cuticle on top of the cells, as many
authors have described. This, however, is nothing else than a
refractive appearance of the cell margin from which the cytoplasm
has slightly retracted. The blood supply is extremely rich.

(A parasitic worm was found in this thyreoid and had caused a
considerable hemorrhage.)

780 J. F. GUDERNATSCH
PSEUDOPLEURONECTES AMERICANUS WALBAUM

Length of specumen 45 cm. The position of the thyreoid gland
in the flounder varies (pl. III, figs. 30, 31). In one ease it formed
a rather compact nodule between the first and second branchial
arteries, While in another the main mass was found in the aortic
bifurcation between and surrounding the first branchial branches
(fig. 20, A). Behind the aortic bifurcation there were only smaller
follicles dorsal and lateral to the aorta. The broad base’of the
deep reaching copula permits only a lateral extension; thus the
thyreoid presents itself as a transverse streak. Small detached
follicles lie close to the base of the gills.

The size of the follicles varies from 15 to 1000u in diameter,
those of about 200u being in the majority. There are also a few
‘giant’ follicles. The epithelial cells of the follicles are closely
arranged and rather high. The nuclei are oval. The blood supply
is rich and lymphatic vessels are well developed.

THYREOID GLAND OF THE TELEOSTS 781

BIBLIOGRAPHY

AnpERSON, O. A. 1894 Zur Kenntnis der Morphologie der Schilddriise. Arch. f.
Anat. u. Phys., Anat. Abt., 177.

BaBeEr, 8. C. 1876 Contributions to the minute anatomy of the thyroid gland
of the dog. Phil. Trans. R. Soc., London, 166, Part 2, 557.

1881 Researches on the minutestructure of the thyroid gland. Phil.
Trans. R. Soc., London, 172, 577.

Boécuat, P. A. 1873 Recherches sur la structure normale des corps thyroide.
Paris.

Borcea, J. 1907 Observations surla musculature branchiostégale des Teleostéens.
Ann. Se. Univ. Jassy, 4, 203.

Cots, F. J. 1905 Notes on Myxine. Anat. Anz., 27, 324.

Cort, C. J. 1906 Das Blutgefissytem des jungen Ammocoetes. Arb. Zool. Inst.
Wien, 16, 217.

Dourn, A. 1885 Studien zur Urgeschichte der Wirbeltiere. Mitt. Zool. Stat.
Neapel.

ErpHEIM, J. 1903 Zur normalen und pathologischen Histologie der Glandula
thyreoidea, Parathyreoidea und Hypophysis. Ziegler’s Beitr. z.
path. Anat. u. allg. Path., 33, 158.

Frereuson, J. S. 1911 The anatomy of the thyreoid gland of Elasmobranchs
with remarks upon the hypobranchial circulation in these fishes.
Am. Jour. Anat., Vol. 11, No. 2.

Forsyty, D. 1908 The comparative anatomy, gross and minute, of the thyroid
and parathyroid glands in mammals and birds. J. Anat. and Phys.,
42, 141, 302.

GaLeorti, G. 1897 Beitrag zur Kenntnis der Secretionserscheinungen in den
Epithelzellen der Schilddriise. Arch. f. mikr. Anat., 48, 305.

GREIL. 1906 Ueber die Entstehung der Kiemendarmderivate von Ceratodus F.
Verh. Anat. Ges., 20. Vers., 115.

GupeErnatscu, J. F. 1909 The structure, distribution and variation of the
thyreoid gland in fish. Am. Ass. Cancer Research, Nov. 27, 1909.
(J. Am. Med. Ass., 54, 227.)

Hirrue, K. 1894 Beitrige zur Kenntnis der Secretionsvorginge in der Schild-
driise. Arch. f.d. ges. Physiol. 65, 1.

JORDAN AND EVERMANN. 1906-00 The fishes of North and Middle America.

Koéuurker, A. 1861 Entwicklungsgeschichte d. Menschen u. d. héheren Tiere,
Leipzig.

LANGENDoRF, O. 1889 Beitriige zur Kenntnis der Schilddriise. Arch. f. d. ges.
Physiol., Suppl., 219.

782 J. F. GUDERNATSCH

Livint. 1902 Organi del sistema timo-tiroideo nella Salamandrina perspicillata.
Arch. It. Anat. Embr., Firenze, 1, 1.

Marcus, H. 1908 Beitrige zur Kenntnis der Gymnophionen. I. Ueber das
Schlundspaltengebiet. Arch. f. mikr. Anat., 71, 695.

Marsuatu, C.F. 1895 Variationin the formof the thyroid gland in man. Jour.
. Anat. and Phys., 29, 234.

Maurer, Fr. 1886 Schilddriise und Thymus der Teleostier. Morph. Jahrb.,
11, 129.

1888 Schilddriise, Thymus und Kiemenreste bei Amphibien.
Morph. Jahrb., 18, 296.

Miuuuer, L. T. 1896 Beitrige zur Histologie der normalen und der erkrankten
Schilddriise. Ziegler’s Beitraige z. path. Anat. u. allg. Path.,19, 127.

Miuuer, W. 1871 Ueber die erste Anlage der Schilddriise und deren Lagebezie-
hung zur ersten Anlage des Herzens bei Amphibien, insbesonders bei
Triton alpestris. Anat. Hefte, 26, 1.

MuvTHMANN, E. 1904 Ueber die erste Anlage der Schilddriise. Anat. Hefte, 26, 1.

PEREMESCHKO. 1867 Ein Beitrag zum Bau der Schilddriise. Zeitschr. f. wiss.
Zool., 17, 279.

Puart, J. 1896. The development of the thyroid gland and of the supraperi-
cardial bodies in Necturus. Anat. Anz., 11.

Scuarrer, J. 1906 Berichtigung, die Schilddriise von Myxine betreffend. Anat.
Anz., 28, 65.

Scumip, E. 1896 Der Secretionsvorgang in der Schilddriise. Arch. f. mikr.
Anat., 47, 181.

Sitvestmer, C. F. 1905 The bloecd-vascular system of the tile-fish, Lopholatilus
chamaeleonticeps. Bull. Bur. Fish. Washington, 24, 87.

Srtmon, J. 1844 On the comparative anatomy of the thyroid gland. Phil. Trans.
R. Soe., London, 134, 295.

StrockarD, Cu. R. 1906 The development of the thyroid gland in Bdellostoma
stouti. Anat. Anz., 29, 91.

STRECKEISEN, A. 1886 Beitrige zur Morphologie der Schildriise. Virchow’s
Arch. f. path. Anat., 103, 131, 215.

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eb iba: * PLATES
py, mee sj

EXPLANATION OF PLATES I TO III

Plates land II show the regions of distribution of the thyreoid elements in
different species. I, aortic bifurcation or first branchial arteries; II, III and IV,
thesecond, third and fourth branchial arteries.

1 Anguilla chrysypa 14 Stenotomus chrysops

2 Clupea harengus 15 Micropogon undulatus

3 Brevoortia tyrannus 16 Tautogolabrus adspersus
4 Osmerus mordax 17 Tautoga onitis

5 Siphostoma fuscum 18 Opsanus tau

6 Fundulus heteroclitus 19 Muraenoides gunellus

7 Fundulus diaphanus 20 Oncorhynchus kisutch
8 Fundulus majalis 21 Salmo mykiss

9 Menidia notata 22 Salmo irideus, age 1 month
10 Sarda sarda 23 Salmo irideus, age 1 year
11 Pomatomus saltatrix 24 Cristivomer namaycush
12 Morone americana 25-27 Salvelinus fontinalis
13. Mugil cephalus

Plate III, Diagrams of actual portions of the thyreoid gland visible to the
naked eye.

28 and 29 Salvelinus fontinalis

30 and 31 Pseudopleuronectes americanus

32-41. Ten specimens of Cynoscion regalis, demonstrating the great variability
in extent and position of the organ within the species.

PLATE I

THE THYREOID GLAND OF THE TELEOSTS

J. F. GUDERNATSCH

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JOURNAL OF MORPHOLOGY, VOL. 21, NO. 4

PLATE II

THE THYREOID GLAND OF THE TELEOSTS

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JOURNAL OF MORPHOLOGY, VOL. 21, NO. 4

THE THYREOID GLAND OF THE TELEOSTS
J. F. GUDERNATSCH

PLATE III

JOURNAL OF MORPHOLOGY, VOL. 21, No. 4

EXPLANATION OF PLATE IV

Plates IV and V, Photographs of the histological features of the thyreoid gland
in different species.

1 Brevoortia. Two follicles with their epithelial cells drawn out into spinous
processes. Dia. 1: 165.

2 Brevoortia. Two follicles, F (the right one containing colloid), and a large
lymph vessel, L, between them. The content of the vessel shows similar droplets
to those sometimes seen on the surface of colloid, and believed by Anderson to
contain the ‘chromophobe’ secretion. Dia. 1:350.

3 Brevoortia. An isolated follicle, F, in the most anterior portion of the thy-
reoid, with neighboring lymph vessels, L. Dia. 1: 160.

4 Brevoortia. General view of the thyreoid in the osseous capsule. Dia.
1:60.

5 Brevoortia. Degenerating epithelial cells and their basal processes. F,
Follicular lumen, FE, Epithelium. Dia. 1: 700.

6 Fundulus heteroclitus. Two pictures showing numerous small capillaries,
Ca, deeply buried in the epithelium, EZ, of the follicles, F. In the lower picture the
epithelium has retracted slightly from the endothelium. A, ventral aorta. Dia.
in a, 1:134; in b, 1:345.

7 Siphostoma. A general view of the posterior end of the thyreoid gland.
Ph, pharynx; A, ventral aorta; F, follicles; Ca, capillaries. Dia. 1: 34.

8 Siphostoma. Single follicles, F, with their colloid forming epithelial cells,
but containing no colloid. Ca, capillaries. Dia. 1: 345.

9 Oncorhynchus. Irregular nuclei, N, of the epithelial cells. H, epithelium of
a follicle viewed from the top. Dia. 1: 650.

: -LATE IV
THE THYREOID GLAND OF THE TELEOSTS PL
J. F. GUDERNATSCH

pce Cana F

Jaches Photo.
JOURNAL OF MORPHOLOGY, VOL, 21, No. 4

EXPLANATION OF PLATE V

10 Salvelinus fontinalis, spec. no. III. Capillary network, Ca, around the
follicles. Note the highly columnar epithelium of the upper and the cuboidal
epithelium of the two lower follicles. Dia. 1: 134.

11 Salvelinus fontinalis, spec. no. II, showing different heights of the epithelial
cells in the same follicle. Dia. 1: 375.

12 Salvelinus fontinalis, spec.no. II. Distribution of the thyreoid elements and
their capillaries, Ca, in the connective and fatty tissue network. Dia. 1: 64.

15 Salvelinus fontinalis, spec. no. III. Much swollen colloid forming cells,
Coz, which have been cast off from the follicular wall into the colloid, Co.
E, epithelium; N, nucleus; V, vesicles in the colloidal substance. Dia. 1: 650.

14 Oncorhynchus. A general view of the thyreoid gland between the first and
second branchial arteries. Ph, epithelial floor of the pharynx; F, follicles surround-
ing the copula; A, ventral Aorta. Dia. 1:60.

15 Anguilla. A duct, D, connecting a small, f, and a large thyreoid follicle, F.
Diaz WelGo:

16 Anguilla. This photograph shows a complex of follicles which, on tracing
through the series, are found to connect with the follicle, F, on the right side of
the illustration. Dia. 1: 170.

17 Cynoscion. A general view of the densely arranged follicles of this species.
Co, colloid; #, epithelium. Dia. 1: 165.

18 Cynoscion. This picture shows the ramifying lymph spaces, L, completely
filled with the same substance as the follicles, F. Dia. 1: 170.

19 Tautoga. The epithelial wall, #, of the follicle, F, is cut somewhat tangen-
tially so that the network of anastomosing capillaries, Va, enclosing the follicles
can be seen. Dia. 1: 145.

20 Clupea. Much swollen colloid forming epithelial cells, Coz, in a colloid
zone. Dia. 1: 170.

21 Sarda. Showing smooth muscle bundles, M, invaded by thyreoid follicles.
Dyan 1260!

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THE THYREOID GLAND OF THE. TELEOSTS or
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Jaches photo.
JOURNAL OF MORPHOLOGY, vot. 21, No. 4

[ Reprinted from THE AMERICAN NATURALIST, Vol. XLIV., July, 1910. ]

ON THE EFFECT OF EXTERNAL CONDITIONS
ON THE REPRODUCTION OF DAPHNIA}

Dr. J. F. MeCLENDON

CorRNELL MepicaL COLLEGE

Since in the great majority of organisms only the
germ cells are capable of reproducing the entire indi-
vidual, the question as to what differences exist between
the germ and body cells, and how they arise, is of gen-
eral interest. Therefore any change of conditions which
affects the germ cells or their relation to the body cells
deserves special study. In Daphnia external conditions
not only affect the relation of the germ cells to the body
cells, but they affect the egg cells in such a manner as to
determine whether they do or do not need fertilization.
The purpose of the present paper is not merely to add
the results of my experiments to those of other investi-
gators, but to tentatively arrange the available data
under a general working hypothesis in the hope that
some more direct method of investigating the relation
of the germ and body cells be devised.

Last spring (March 10) I began experiments on the
effects of environment on Daphnia pulex, De Geer, with-
out knowing that Woltereck was working on the same
line. The material came from a small pool investigated
by Dr. W. C. Curtis and containing a single strain of
this and no other species of Daphnia. For the first few
weeks some ice remained on the pool and the tempera-
ture did not much exceed 4° C.; after this it rose steadily
to about 20° by the end of May. Specimens from the
pool were examined at intervals as a control on the ex-
periments.?

* From the Zoological Laboratory of the University of Missouri and the
Histological Laboratory of Cornell University Medical College, New York
City.

* When the daphnids were crowded in dishes of the same pool water they
soon began to die, owing to the accumulation of their excretions. When

404

405 THE AMERICAN NATURALIST [Vou. XLIV

EFFECT OF ENVIRONMENT ON DIFFERENTIAL GROWTH

By differential growth I mean the unequal growth of
different parts, viz., the germ and body cells. Only
parthenogenetic females were used, and each was kept
separately in the same quantity of water. All measure-
ments were made at sexual maturity, 7. e., when the
first eggs appeared in the brood pouch. Warren*® found
that under uniform conditions there was a slight varia-
bility, but Woltereck showed that these fluctuating varia-
tions were very small, though he did find mutations as
rare occurrences.

Nutrition.—l had in the laboratory a pure culture of
a unicellular green alga which the daphnids ate readily.
This alga did not remain entirely suspended in the water,
but as the daphnids fed on the bottom as well as while
swimming, and stirred up the alge, it can not be said
that most of the food was out of their reach.

Those with a superabundance of food were larger at
sexual maturity and had a shorter spine than those with
insufficient food, and conversely. The smaller size and
longer spine of the starved daphnids are characteristic
of immature stages.

Temperature—tThree sets of experiments were
transferred suddenly to artesian tap water many died, though with a
gradual change all lived. The composition of the tap water was as follows:
Ca, .148 X 10° molecular; Mg, .1 X 10 molecular; CO,, .045 X 10 molec-
ular; SO,, .146 X 10° molecular; Cl, .055 x 10-§ molecular. Besides these
were very small quantities of silica, clay, iron, ammonia and nitrates, and
traces of lithium and potassium. Beside the carbonates the water when
drawn from the tap was super-saturated with carbon dioxide. In order to
find the cause of death from change of water I added various amounts of
molecular solutions of certain salts. The toxicity of cations increased as
follows: NA<1/2Ca<K, and of anions: Cl<1/2S0,<HCO,;<1/2 HPO,.
But as K became toxie only on 1/100 and HPO, on 1/500 molecular concen-
tration there must be other toxic substances than salts in the water. The
carbon dioxide in the water killed crayfish and was probably the most toxic
constituent to the daphnids.

***An Observation on Inheritance in Parthenogenesis,’’ Proc. Roy. Soc.

London, 1899, LXV, and ‘‘On the Reactions of Daphnia magna to certain
changes in Environment,’’ Quart. Jour. Mier. Sc., 1900.

No. 523] REPRODUCTION OF DAPHNIA 406

started: at 4-10°, 19-20° and 29-31° C. The results
were as follows:

iA Body L _|Ratio of Average Spine’ Time from Hatching
| yersee Body ever Length to Body Length. to Sexual Maturity.

4-10° 22 0.24

35 days
19-21° 20 0..5 | ie
29-31° 17.7 | 0.27 pS

1 unit = millimeters.

All were given a surplus of food daily. It may be
observed that a higher temperature has the same effect
as insufficient food.

Salts——Since salts have such a marked effect on the
development of marine and some fresh-water animals,
I placed daphnids in the strongest solutions of various
salts that they would live in (without acclimatization).
The effects in two months (four generations) were un-
noticeable. .

Light.—Cultures were kept in the dark and in diffuse
and direct sunlight, but no effect was observed.

A number of observers have recorded season-poly-
morphism in daphnids. Wesenberg-Lund?t pointed out
that when the specific gravity and consequent buoyancy
of the water decreased—by heat in summer—the body
of the daphnids became smaller or were provided with
outgrowths, so as to offer a greater resistance to sinking.
Wolfgang Ostwald® produced, all at the same time, the
forms that occurred in nature at different seasons, by
varying the temperature. He emphasized the fact that
rise in temperature lowered the internal viscosity of the
water. He found that in the warm cultures the daphnids
often became productive at an undeveloped stage and,
as is true generally, reproduction retarded body growth.

*“¢Ueber das Abhingigkeitsverhiltnis zwischen den Bau der Plankton-
organismen und den specifischen Gewicht des Siisswassers,’’ Biol. Centlb.,
1900, XX, pp. 606-619, 644-656, and ‘‘Studier over de Danske siers
Plankton,’’ Copenhagen, 1904.

5¢¢ Experimentelle Untersuchungen tiber den Saisonpolymorphismus bei
Daphniden,’’ Archiv f. Entwicklungsmech. d. Organismen, 1904, XVII.
p. 415.

407 THE AMERICAN NATURALIST [ Vou. XLIV

Woltereck® maintains that Ostwald’s results were due
to the fact that at a higher temperature the daphnids
need more food. Woltereck caused decrease in body
length both by starving and by increasing the tempera-
ture, but the latter was not effective with an optimum
supply of food. He found that more food than the
optimuin produced effects similar to starving. Raising
the internal viscosity of the water by adding quince gum
produced no effect. He showed that though feeding in-
fluenced the differential growth, there was a cyclical
tendeney for this to vary, viz., season-polymorphism.
However, the effects of prolonged abundant feeding were
inherited to some degree.

One might interpret these results in different ways.
It is known that the temperature coefficients for various
chemical reactions are slightly different. Possibly the
mean of the temperature coefficients for the processes
in the development of the reproductive organs is higher
than the same for the body wall, and at a higher tem-
perature the germ cells would develop faster. However,
under adverse conditions the ‘‘affinity’’ of the repro-
ductive organs for nutriment is greater than that of the
rest of the body, so with deficient food the body wall is
retarded more than the germ cells in development. The
higher temperature may be considered an adverse condi-
tion since the mortality is greater. In this way starv-
ing has the same effect as a higher temperature.

Langerhans? found that accumulation of excretions
caused shortening of the spine in daphnids. I do not
know what relation this bears to the above results.

°**Ueber naturliche und kunstliche Varietatenbildung bei Daphniden,’’
Verh. Deutsch. Zool. Gesell., 1908, p. 234; and ‘‘ Weitere experimentelle
Untersuchungen iiber Artveranderung, speziell tiher das Wesen quantitativer
Artunterschiede bei Daphniden,’’ ibid., 1909, p. 110.

““«Ueber experimentelle Untersuchungen zu Fragen der Fortpflanzung,

Variation und Vererbung bei Daphniden,’’ Verh. Deutsch. Zool. Gesell.,
1909, p. 281.

No. 523] REPRODUCTION OF DAPHNIA 408

EFFECT OF EINVIRONMENT ON THE LiFE CYCLE

In most species of daphnids, generations of partheno-
genetic females alternate with generations of males and
females which produce eggs that must be fertilized, and
either frozen, dried or kept a long time before they will
develop (resting or ‘‘winter’’ eggs). In different
species the number of successive parthenogenetic gen-
erations varies. In some all are, and in some none are,
parthenogenetic.

I found that heat hastened the appearance of sexual
forms, as did starving or the accumulation of excretory
products. All of these factors might be combined in the
drying up of a pond, as heat would aid in drying, and
drying would concentrate the daphnids and their ex-
eretions, and concentration of the daphnids would cause
them to eat up the alge faster than they could multiply.
However, by keeping the culture cold, fresh or well-fed,
or all combined, I could delay but not prevent the ap-
pearance of sexual forms.

Kurz’ said the drying up of the water caused the ap-
pearance of sexual forms, and Schmankewitz® suggested
that it was the increase in salts. Weismann! tested
both of these hypotheses and concluded that they were
wrong. He also tried the effect of food and tempera-
ture, with varying results. He concluded that the life
eycle was fixed for each species and variety. Issako-
witz!" concluded that cold favored the appearance of
sexual forms and warmth favored the parthenogenetic.
Also, hunger favored the appearance of sexual forms
and abundant food the parthenogenetic. It may be that
cold retarded multiplication of the food plant or the

8¢¢Todekas neuer Cladoceren nebst einer kurzen tibersicht der Cladoceren-
fauna Bohmens,’’ Sitz. Ber. math. naturw. Wien, 1875.

®*“Zur Kenntnis des Einflusses der ausseren Lebensbedingungen auf die
Organisation der Tiere,’’ Zeit. wiss. Zool., 1877, XXIX.

70*<Beitrige zur Naturgeschichte der Daphnoiden, VII,’’ Zeits. wiss.
Zool., XXXTII, p. 111.

*<*Geschlechthestimmende Ursachen bei den Daphniden,’’ Biol. Cen-
tralb., 1909, XXV, pp. 529-536.

409 THE AMERICAN NATURALIST [ Vou. XLIV

movements of the daphnids so that they did not keep
the alge stirred up in the water sufficiently to get at
them. The parthenogenetic egg arises from four cells,
but a large number of cells enter into the composi-
tion of the fertilizable egg. If the latter egg is not
fertilized it is absorbed and, as Issakowitsch noted, fur-
nishes food for the development of parthenogenetic
eggs.

Woltereck'? found that starving hastened the appear-
ance of sexual reproduction, but a concentration of food
above the optimum produced results similar to starving.
He found, as Weismann maintained, a cyclical tendency
toward the alternation of sexual and parthenogenetic
generations which, contrary to Weismann, was tempo-
rarily influenced by nutrition, and the effects of constant
nutrition over a long period was inherited to some ex-
tent.

Langerhans’? found that the accumulation of exere-
tions caused the decrease in numbers of parthenogenetic
females in the autumn and thinks that the appearance
of sexual forms is due to the same cause.

The life eyele of a daphnid is, therefore, an hereditary
tendency, but can be influenced by nutrition and probably
by temperature and the accumulation of excretions.
Nutrition is the most important factor, and former ex-
periments on the effect of temperature and the drying
up of the water were complicated by secondary effects
on concentration of food and excretory products.

Discussion of Results—Two views might be held as
to the origin of the differences between the germ and
body cells: the differences might be the result of differ-
ence in position in the embryo, or of unequal mitoses.
In the parasitic copepods I found the primary germ cell
to arise by an unequal mitosis at the fifth cleavage of
the ege. The germ cell when first formed is one thirty-
second of the total number of cells, but owing to the more

2 0c. Cit.
Loc cit

No. 523) REPRODUCTION OF DAPHNIA 410

rapid division of the body cells this ratio decreases. In
fact the chief difference between the yolk-free body cells
and the (yolk-free) germ cells is the slow rate of division
of the latter. Finally, the eggs will not divide at all un-
less specially stimulated, by fertilization. The question
now arises: what causes the cell to divide. Sacks found
that plant cells divide when they have reached a certain
size. This rule has been extended to animals, and the
final size of the cell found to be determined by the ratio
of nucleus to cytoplasm. This rule may apply to the
germ cells, since it appears that after the egg cell, pri-
mary oocyte, reaches a certain size any additional food
absorbed does not cause growth of the protoplasm, but
is precipitated as yolk.

If the egg is properly stimulated, rapid growth of
protoplasm and cell divisions follow. From the study
of artificial parthenogenesis it appears probable that
stimuli which lead to development of the egg increases
the permeability of its plasma membrane. If this be
true we may say that the germ cells are distinguished
by the fact that their plasma membranes are poorly
permeable and retard those reactions between the cell
contents and environment which lead to growth and cell
division. In other words, the optimum intensity of stim-
ulation toward growth and division is higher for the
germ cells than for the body cells.

This difference is probably due to a difference in the
colloids of the cell, which in animals could be explained
as the result of an unequal mitosis. This explanation
may be modified so as to apply to plants. Klebs has
shown that those conditions which are adverse to vege-
tative growth of plants (too strong stimuli?) call forth
flowers. Perhaps there are slight differences in the
sensitiveness of plant cells to stimuli, and as the stimuli
increase, those initially least sensitive cells acquire
further immunity to the stimulus, whereas those initially
more sensitive cells are overstimulated and weakened.
Thus the difference between germ and body cells is grad-

411 THE AMERICAN NATURALIST [Vou. XLIV

ually aequired. The Malpighian layer of the skin may
be stimulated to proliferate more rapidly, but if the
stimuli are too strong the growth will be retarded instead
of increased. On gradually increasing the stimulus im-
munity to it may be acquired.

To apply this hypothesis to the daphnids: conditions
which are adverse to the growth of the body cells, such °
as extremes of temperature (viz., high temperatures) or
of concentration of excretory products, or disorded nu-
trition, either fail to retard the development of the germ
cells or stimulate their development, so that in either
case the daphnid becomes sexually mature at a less de-
veloped stage. Under the less extreme conditions the
eges develop on receiving the slight stimulus incident to
their transfer to the brood pouch, but under the more
extreme conditions those eggs which develop at all must
be stimulated by fertilization before they develop.
These two types of eggs may perhaps develop from two
kinds of cells, or the sexual egg may arise from the same
kind of cell producing the parthenogenetic egg by ac-
quiring an immunity to slight stimuli. Whereas more
than one cell goes to make up a single egg; only one
nucleus is retained, and it may be said that one cell is the
ege cell and the remainder furnish its food.

’

THE DEVELOPMENT OF ISOLATED BLASTOMERES
OF THE FROG’S EGG

J. F. McCLENDON

From the Zoélogical Laboratory of the University of Missouri and the Histological
Laboratory of Cornell University Medical College, New York City

WITH TWO FIGURES

After the numerous proofs furnished by various investigators,
there is no question that differentiation begins very early in
development, but the determination of the exact stage at which
various differentiations begin, has been hampered by technical
difficulties. The importance of overcoming these difficulties lies
in the fact that it is only by an exact study of the early develop-
ment that we can ever hope to know the mechanics of differen-
tiation. The later stages are so complex that it is doubtful that
they could be analysed even though the early stages were under-
stood.

The frog’s egg, owing to its large size, has been a favorable
object of study. O. Hertwig,! after pricking one blastomere,
found that a complete embryo was formed.

Roux,? in similar experiments obtained a half embryo from the
uninjured blastomere, and explains Hertwig’s observation by the
fact that some of these half embryos became complete larvze
by a process of regulation which he called post-generation. This
might occur in the following different ways: first, the half embryo
might fold together on the side corresponding to the median plane
and be transformed into a whole embryo of half size; second, the
injured blastomere might recover from the operation and develop
into the missing half; and third, the injured blastomere might be

"Arch. f. Mikroscopische Anat., 1893, XLII, p. 662.
"Ges. Abh. zur Entwicklungsmechanik der organismen, Leipzig, 1895.

426 J. F. McClendon.

reorganized under the influence of the half embryo into the miss-
ing half. The occurrence of post-generation, and especially the
third type, has been denied by a number of investigators who
repeated this experiment. Laquer? has reinvestigated the ques-
tion.

This production of a half embryo from one blastomere is not
due to the inability of the egg to produce more than one embryo,
since Schultze produced double forms by inverting the egg in the
two-cell stage, and Morgan® produced a complete embryo from
one blastomere by pricking the other blastomere and then invert-
ing the egg. In order to determine whether this inversion of the
egg and consequent stirring up of its contents is necessary for the
production of a complete embryo from one blastomere, I attempt-
ed to find a frog’s egg that would permit the complete removal of
one blastomere without death of the egg. No one has hitherto
been able to do this, although Roux® observed a partial but very
abnormal development of extra-ovates of the frog’s egg.

Last spring at Columbia, Missouri, I located the breeding
places of Rana pipiens and Chorophilus triseriatus. The unseg-
mented eggs of both species could be collected in the ponds and
small streams, or in the frog cages in the laboratory, the eggs of
the latter species being more easily obtained.

I tried various methods of removing one blastomere in the two-
cell stage. It was possible to suck out one blastomere with a
capillary pipette connected with a rubber tube, one end of which
was held in the mouth, but it was very difficult to pierce the jelly
with the pipette without killing the egg, and the suction often
removed both blastomeres. A better way of making a hole in the
jelly was the time honored method of piercing it with a hot
needle. A very coarse needle was used and heated to a tempera-
ture that would coagulate a portion of the blastomere into which
it was thrust. On withdrawing the needle, the coagulated parts of
the jelly and blastomere were pulled out, thus leaving a large hole

“Arch. f. Entwicklungs. 1909, XXVIII, p. 327.

‘Arch. f. Entwicklungsmech., I. p. 298.

®Anat. Anz. 1895) DG pees:

*Jahresbericht d. Schles. Ges. f. vaterl. Cultur, Juni, 1887.

Isolated Blastomeres of the Frog’s Egg. 427

through which the remaining part of the blastomere could be
extracted. The eggs of Chorophilus were more easily handled,
since the jelly was softer and the egg itself did not collapse readily.
This egg is not so small as to make operations difficult with the
low power of the binocular microscope.

When the first cleavage furrow had extended almost around the
ege, the greater part of the jelly was carefully removed with filter
paper and the puncture made. The egg was then allowed to rest
until the first cleavage plane had completely divided it. At this
time the remains of the punctured blastomere were removed
with the capillary pipette. In case the last remnants could not be
removed, the egg was allowed to rest again until the remnants of
the punctured blastomere turned pale, which was an indication that
they were more brittle and could now beremoved with a fine needle.

When the egg was punctured, the perivitelline space was oblit-
erated by the escape of the contained fluid and the jelly pressed
close to the egg, an opening was also made through which bacteria
might enter; therefore, the eggs laid in the laboratory under a
more sterile condition, gave better results than those laid in the
ponds. Care was taken to keep the egg normally orientated in
reference to the direction of gravity both during and after the
operation.

The operated eggs were observed with the binocular micro-
scope and the early cleavage noted. The gastrulation was watched
through the sides and bottom of the glass dishes in which the eggs
were kept. After the embryos became ciliated, some of them
turned in a normal manner so that they could be observed in all
aspects, from above.

A large number of eggs of both Rana pipiens and Chorophilus
triseriatus were operated on. All of the former died, but of the
latter a considerable number gastrulated and several reached the
tadpole stage. Two were four days, three were five days and one
was eight days old when preserved for sectioning.

The control eggs hatched in less than eight days, but the operat-
ed eges were retarded in development and the resulting tadpoles
were too weak to break through the jelly, although the twitching
of their tails showed that the muscles were functional.

428 J. F. McClendon.

The development of the operated eggs was very similar to that
of the normal, and quite different from that of the uninjured blasto-
meres in Roux’s experiment. The remaining blastomere rounded
up gradually after its partner had been removed, and the blastula,
gastrula and later stages were wholes of half size. As the mortality
was great, it is to be expected that defects would be observed in
many of the specimens, but these defective specimens could not
be interpreted as half embryos. The only defect that I could
observe in the oldest specimen was the small size of the suckers,

Fig. 1 Fig. 2

Fig. 1. Chorophilus triseriatus: a section of the left eye of a tadpole developed
from one of the first two blastomeres.

Fig. 2. A section of the right eye of the same tadpole as in fig. 1.

before the pericardium began to swell so that the heart beats
could no longer be observed.

Sections confirmed the observations on the living material
and showed that the embryos were complete and not half-
forms.

Figs. 1 and 2 represent the right and left eyes seen in transverse
sections of the eight day larva. The right eye lacks a lens, and
the left eye is not quite normal, yet these defects make no ap-
proach to those of a half-larva since they are not very extensive

\ ellie’

Isolated Blastomeres of the Frog’s Egg. 429

and are not all on the same side. There is some displaced pigment
in the center of the lens, and the cells of the optic cup have not
preserved their normal arrangement. An examination of the
ectoderm showed more or less disintegration, and the defects
observed may be explained by the fact that the specimen was
dying when it was taken out of the water to be preserved.

These differences in the two eyes of the eighty-day embryo
from one blastomere, are the most striking differences between
the right and left sides. The ear vesicles are almost identical on
the two sides, and the same is true of the other paired organs.
Furthermore, no general defect of the anterior, posterior, dorsal
or ventral regions of the body could be found. The study of the
five other embryos developed from operated eggs, and cut in
serial sections also revealed complete forms.

Roux described right and left ‘“‘hemiembryones laterales”’ and
also ‘‘hemiembryones anteriores”? developed from one _ blasto-
mere in the experiments mentioned above.

Since-the anterior end of the neural folds lies on one side of the
egg between the equator and the animal pole, i. e., about the
region in which the gray crescent appeared, and the dorsal side
of the embryo is formed chiefly on this side, it is probable that in
case the first cleavage plane were transverse to the plane of sym-
metry, it would divide the egg into the halves that would form
the dorsal and ventral parts of the embryo respectively. There-
fore the ‘“‘hemiembryones anteriores’? might better be described
as ‘“‘dorsales” as indicated by Przibram. Roux obtained no
“‘posteriores”’ (‘‘ventrales”’ of Przibram) and since a number of
the eggs died, it is probable that those blastomeres which corre-
spond to the ventral part of the embryo, and therefore contain no
part of the gray crescent, are not so well adapted to independent
development. In my experiments the position of the grey crescent
as marking the future dorso-anterior region of the embryo was
not recorded, but it seems probable from the results of Roux’s
experiment, that those isolated blastomeres containing no part of
the grey crescent did not develop.

As noted above, Roux found that half-embryos sometimes be-
came complete embryos by post-generation.

430 J. F. McClendon.

None of the three types of post-generation occurred in my
experiments. The isolated blastomeres rounded up in a manner
that might be compared to the first type of post-generation, but
this regulation occurred before an erabryo was formed, and might
be distinguished as ‘“‘immediate. ”’

It appears then that the egg at the time of the first cleavage is
differentiated in the direction of the primary axis, and further-
more in the direction of a plane passing through this axis and bi-
secting the grey crescent, so that the egg may be divided in this
plane into two totipotent halves. The halves of the egg divided
in any other plane may or may not be totipotent. The formation
of a half embryo instead of a whole embryo from one blastomere
of the two-cell stage is due to the presence and mechanical inter-
ference of the other blastomere.

In this connection may be mentioned the results of Endres,
Herlitzka and Spemann on the development of the first two blasto-
meres of urodeles when separated by constriction with a fine hair.
If the constriction was slight, the hair merely marked the plane
of the first cleavage, if it was deeper, double monsters occurred,
and if it was complete, two perfect embryos resulte1.

In from 66 per cent to 75 per cent of the eggs, the first cleavage
plane became a frontal plane of the embryo, and in the remainder,
it became the median plane. In the latter case complete constric-
tion gave rise to two complete embryos, but in the former case,
complete constriction resulted in one complete embryo developed
from the prospectively dorsal half of the egg, whereas the pros-
pectively ventral half did not develop beyond gastrulation. Thus
it appears that the eggs of the urodeles possess the same organi-
zation as the eggs of the anura, with the difference that the first
’ cleavage plane is usually frontal in the former, and median in the
latter in relation to the embryonic axes.

Accepted by The Wistar Institute of Anatomy and Biology, January 19, 1910. Printed June 21, 1910.

On the Effect of Centrifugal Force on the Frog’s Egg.
By
J. F. McClendon

(From the Histological Laboratory of Cornell University Medical College, New York City, U.S.A.)

With 9 figures in the text.

The centrifuge has become a popular piece of apparatus for the
analysis of the structure and developmental processes in the animal egg.
The results thus obtained are very interesting, but the interpretation of
these results have been divergent in some cases. Therefore it may be
of interest to consider certain facts that have a bearing on the disputed
points.

On the Cytoplasmic Structure.

In a recent paper, Gurwitscu (09) maintains that centrifugal force
destroys the alveolar structure of the frog’s egg by separating the con-
tents of the alveoles, enchylemma, as a layer at the animal pole, from
the substance forming the walls of the alveoles, hyaloplasm, which then
forms a layer just below the enchylemma. GurwitscH had reported
the observation before the Anatomische Gesellschaft at Jena in 1904,
and similar results were published by Konopacka (08), with this diffe-
rence that Gurwirscu, following the alveolar theory of Burscutt, called
the substance that rises to the top of the egg enchylemma, whereas Kono-
PACKA, adhering to the reticular theory of Leypia used the term hyalo-
plasm to denote the same substance. In order to throw light on the
question whether the alveolar structure is destroyed by centrifugal force
I have made a study of the cytoplasm of the normal egg.

A small portion of a thin smear of the living ovarian egg of Rana
pipiens is shown in fig. la. The large ovals represent yolk-platelets, the
black dots pigment, and the small circles represent spheres of a sub-

386 J. F. McClendon

stance that can hardly be differentiated in the living egg from the sub-
stance of the yolk-platelets. Fig. 1b shows these bodies in an ege fixed
in osmic acid. The small circles are here striated to denote that they re-
present bodies that take the characteristic osmic impregnation for fat.
There is some difference in the size of these fat droplets in the living cyto-
plasm and the osmicated section, but none of them in either preparation
are as large as the yolk-platelets. If a smear of the living ege be fixed in
osmic¢ acid and immediately examined, some of the fat droplets are seen
in the act of fusing to form larger drops, fig. le. By treating a smear

Fig. 1. Fig. 2.

..

Cytoplasma of ripe ovarian egg of Rana pipiens;
a. living egg burst under cover-glass; b. section
fixed in osmic; ¢. fresh smear fixed in formal-
dehyde vapor and stained in alcoholic solution

of Sudan III. The large ovals are yolk-platelets; Juncture of the fatty layer(above)

the black dots pigment and the striated bodies and protoplasmic layer (below)

gave the reaction for fat; the small circles in in the cytoplasm of the egg of

a. represent fat, this is also true of the small Chovophilus trisertatus centri-

circles in d. which represent fat droplets which fuged in the four-cell stage,

are too small to show a perceptible intensity centrifugal force = 2771 gra-
of stain. vity for seven minutes.

of the living egg, fixed for a few seconds in formaldehyde vapor, with an
alcoholic solution of Sudan III, the fat droplets are stained, but they
vary much more in size than in any of the above preparations, some of
them being larger than the yolk-platelets, fig. 1d. I attribute this varia-
tion in size of the fat droplets in different preparations to the fusion of
the original droplets to form larger drops, a process which is due to the
rubbing of the smears or to the addition of substances which reduce the
surface tension of the droplets. This last point is illustrated by the fact
that if the osmicated smear be treated with the alcoholic solution of
Sudan III, the fat droplets may be seen to fuse and form larger drops.
3y chemical analysis I found (09, 1 and 3) the pigment granules to
be composed of a melanin containig 0,6°% of sulphur and 9% of nitrogen;

et a

On the Effect of Centrifugal Force on the Frog’s Egg. 387

the yolk-platelets to be composed of 6°, of lecithin and 94°, of batra-
chiolin, a nucleo-albumin containing 1,2°/, of phosphorus, 1,3°/, of sulphur
and 15°, of nitrogen; and the fat droplets to be composed of a liquid fat,
a solid fat melting at 58 degrees, and a yellow lipochrome. ‘
In the living frog’s egg, I have not been able to observe any struc-
ture to the cytoplasm proper, that is, to the substance that fills the spaces
between the yolk-platelets, fat droplets, and pigment granules. Gur-
witscH (09) supposed an alveolar structure to exist, but which was ob-
scured by the yolk-platelets. I examined younger ovarian eggs, contai-
ning no yolk, fat or pigment, alive under a 2 mm oil immersion lens and
could observe no structure in the cytoplasm except clumps of granules
in certain regions, the yolk-nuclei. In a former paper (09, 1) in speaking

Fig. 3. Fig. 4.

The same structure as in fig. 2,
in the ovarian egg of the toad,
centrifugal force = 2771 X gravity
for two hours. The greater visco-
sity of the ovarian egg necessitated
a longer duration of the force to
obtain the samé stratification.

Portions of cytoplasm in sections
of ovarian eggs of Kana pipiens
containing no yolk.

- of the alveolar structure of the normal frog’s egg, | referred to the spaces

DD?

filled with yolk-platelets and fat droplets and containing in addition en-
ehylemma, as I supposed. It is hardly probable that spaces entirely occu-
pied by yolk or fat can be rightly called alveoles in Burscuit'’s sense.
If the frog’s egg be centrifuged, I observed (09,1) that the fat dro-
plets rise to the top of the egg and fuse to form larger drops, whereas the
cytoplasm proper forms a layer just beneath the layer of fat drops. A
small portion of a section showing a part of each of these two layers is
represented in fig. 2. Each layer has an alveolar or reticular appearence,
the meshes in the fatty layer being larger than those in the layer below,
which Gurwirscu calls artefacts. The meshes in the fatty layer are
formed of the coagulated cytoplasm that separated the fat drops until
the latter were dissolved out with xylol. The meshes in the layer below
are probably artefacts as GuRwiTscH maintains, but they are about as

large as the meshes seen in the cytoplasm of fixed ovarian eggs contai-

388 J. F. MeClendon

ning no yolk, fig. 4, and if all these are artefacts, the assertion that an
alveolar structure in Burscutt’s sense exists in the cytoplasm of the frog’s
egg is not based on optical evidence (cf. RuumMBLER). The fact remains
that the spaces which Gurwirscn supposed to be filled with enchylemma
in the centrifuged frog’s egg are in reality filled with fat droplets, and the
process which he describes as a destruction of the alveolar structure is in
reality an aggregation of the fat droplets. Whether the process which
GurwitscH describes as a restitution of the alveolar structure, which
occurs in eggs after they have been removed from the centrifuge, is a
redistribution of the fat droplets, I cannot positively affirm, as I have
not determined that the fat droplets are ever uniformly redistributed,
neither do Konopacka’s figs. 12and14, Pl. XXVI indicate such a process,
and although on p. 772 she states that the vacuolated layer seen in cen-
trifuged eggs disappears soon after the eggs are removed from the centri-
fuge, she adds that the stratified arrangement of substances persists
during the entire development of the eggs.

On the Cause of the Abnormal Development of Centrifuged Eggs.

GuRWITSCH assumes that the abnormal development of centrifuged
frog’s eggs is due to the failure of the nuclei to fit the portions of the cyto-
plasm which surround them. This he attributes to the meniscus for-
mation, which did not occur in my experiments. The question arises
whether such an assumption is necessary to explain the facts. I con-
cluded (09, 1) that the physical and chemical differences between the
layers of the egg were sufficient to account for the abnormal develop-
ment, and found that if the centrifugal force was sufficiently prolonged
(2771 x gravity for 20 minutes) not even the beginning of segmentation
ever occured. GuRwiTscH maintains that similar explanations are in-
validated by the fact that sometimes a portion of one layer will segment
while the remainder of the layer is unsegmented, that is to say that the
injury to the egg is independent of the stratification. It should be re-
membered that frog’s eggs are easily injured by handling, and that cen-
trifugal force not only stratifies the ege, but causes currents in the egg
and presses the eggs one against the other The currents in the egg are
indicated by the streaming of the black pigment observed by Morcan
and myself. PrruGER observed that if the frog’s egg be inverted, the
small cells appear in the white hemisphere, and Born demonstrated this
to be accompanied by an inversion of the egg contents. It has been pro-
ved that the polarity of the frog’s egg is determined by the arrangement
of egg substance, either deutoplasm or protoplasm proper. The nearly

.

On the Effect of Centrifugal Force on the Frog’s Egg. 389

normal development of certain centrifuged eggs has been used as an ar-
eument for the position that the deutoplasm moved by the centrifuge
has little or no effect on polarity. If this be true the polarity is determi-

Fig. 5.

Section through the equator of a mitotic figure

in an egg of Rana pipiens that was centrifuged

in the two cell stage, centrifugal force =

277i X gravity for one minute. The yolk

platelets and pigment granules, in being preci-

pitated, were stopped by the spindle and piled
up on it.

Fig. 6. .

Group of chromosomal vesicles surrounded by
four asters in a horizontal section of the egg
of Chorophilus trisertatus that was placed in
the centrifuge in the four cell stage, centri-
fugal force = 565 gravity for 45 minutes.

ned by the protoplasm. If the polarity be determined by the cytoplasm
alone, currents might destroy the relation of the polar axes of various

x

\

From a section of the same egg shown in fig. 6, showing a nucleus, one of whose asters has apparently
divided and one of the products of.the division has moved away.

regions to one another, just as would be the case if a magnet were melted
and its substance stirred.

Another cause for the abnormal development of centrifuged frog's
eggs might be abnormalities in the mitotic figures. I have previously

390 J. F. McClendon

described (09, 1) the flattening of the mitotic figures in the direction of
the centrifugal force. In some cases the yolk and pigment is piled up upon
the spindle, as shown in fig. 5, which represents a cross section of the spindle
containing the equatorial plate. The longer the eggs remain in the centri-

Two groups of chromosomal vesicles, abnormal or incipient nuclei, indicating division of nuclei without
cytoplasmic division, in an oblique section very near the animal pole of an egg of Rana pipiens that
was placed in the centrifuge in the eight cell stage, centrifugal force = 565 > gravity for 4/2 hours.

fuge the greater the abnormalities in the mitotic figures. Supernumerary
asters appear, that are probably the products of the division of the asters
of the mitotic figures. Fig. 6 represents the chromosomal vesicles sur-

Fig. 9.
AYA SS
Ser —~\

F Y
————_—BHi7,

eg Z SS Ly
WiFi j NN
N\) GfZEM
SA
A
SSS =<

\ =— — =
——SS —

\ SSS
SSS

7i\N

<j

From a section lower down, in the egg shown in fig. 8, representing numerous asters in division and
far removed from any chromatin.

rounded by four asters in an egg centrifuged 45 minutes. Fig. 7, from
the same egg, represents a nucleus with two asters, but nearby is a third
aster which is apparently a product of the division of one of the asters
connected with the nucleus. Fig. 8 shows two masses of chromosomal

On the Effect of Centrifugal Force on the Frog’s Egg. 391

vesicles surrounded by numerous astral radiations in an egg centrifuged
one and a half hours. Fig. 9, from a region of the same egg further re-
moved from the animal pole, represents numerous asters apparently in
the act of division and not connected with chromosomes or nuclei. Du-
ring the action of the centrifuge the nuclei remain very near the animal
pole, though mitotic figures and supernumerary asters may be found a
little further down in the egg. If the centrifugal force is sufficiently
strong, division of the nuclei without division of the cytoplasm occurs,
which may or may not be followed by division of the cytoplasm after
the eggs are removed from the centrifuge. Just what effect these ab-
normalities in the process of mitosis have on the development of the em-
bryo, I cannot determine, but it is probable that they are partly respon-
sible for the abnormalities in development.

In conclusion to this section, it seems probable that the abnormal
development of centrifuged frog’s eggs may be due to one of a number of
causes or to a combination of causes. Currents in the egg may be partly
responsible for its abnormal development, which possibility is rendered
more probable by the fact that the egg of Arbacia, in which centrifugal
force does not produce currents, is little effected in its development
thereby, however I have given other reasons for this difference (09, 2).

On the Effect of Centrifuga! Force on the Egg at Different Stages of
its Development.

It has been recorded by several observers that the degree of the
effect of centrifugal force on the frog’s egg at different periods of its deve-
lopment is different. Konopacka studied this subject systematically,
and found that the ratio of abnormal to normal embryos resulting, in-

- ereased markedly when the force was applied after the entrance of the
: spermatozoan, and that there was a second period of increase of this
_ Tatio when the force was applied just before the completion of the third
cleavage, after which the ratio decreased again. Without knowing of
Konopacka’s results, in the spring of 1908, | performed similar experi-
ments, which might be of interest since the centrifugal force was greater
_ and acted for a shorter time (1 minute) and the results were denoted by
_ the character of the most perfect embryos. The eggs of Rana pipiens
_ laid by one female were divided into lots, and at the end of each ten
minute period one lot was centrifuged with a force equal to 2771 x gravity
for one minute. Part of each lot was then preserved for sectioning and
the remainder placed in a dish of tap water to await depelopment. The
highest developmental stages reached by the eggs of the various lots

392 J. F. McClendon

were classified as normal tadpoles (except in coloration), abnormal tad-
poles, partial embryos and irregular cleavage stages. In the list below,
the numbers refer to the succession of ten minute periods:

1 normal tadpoles

» »)

bo

10 » »

11 irregular cleavage (@ pro-nucleus seen in sections)
12 ne »

13 normal tadpoles

Commencement of first cleavage furrow
14 normal tadpoles

15 irregular cleavage

16 normal tadpoles

17 partial embryos

18 normal tadpoles

19 » »

Commencement of second cleavage furrow
20 normal tadpoles

21 partial embryos

22 » »

Commencement of third cleavage furrow

24 abnormal embryos

25 irregular cleavage.

In a second set of experiments, lots corresponding to lots 20—25 in
the above list all gave partial embryos as the best results. Although
the results are not quite uniform, it appears that the early stages after
the eggs are laid, as indicated by the first ten lots, are less affected by
centrifugal force than later stages. I attribute this to the fact that the egg
is more viscid in early stages and consequently the stratification of sub-
Stances and production of currents is less than in later stages. Thus I
found that the time required to stratify the ripe ovarian egg by means
of a given centrifugal force to be much greater than that required during

On the Effect of Centrifugal Force on the Frog’s Egg. 393

early cleavage. The same is true for the ovarian egg of the toad, fig. 3.
With closely consecutive stages it is much more difficult to make quanti-
tative comparisons.

BraTaszewicz found that the frog’s egg increased in volume pro-,
gressively after it was placed in water (except while the perivitelline
space was forming) and that this occured whether it were fertilized or not.
In this connection might be mentioned the fact recorded by Backman
and Runpstr6 that the osmotic pressure of the frog’s egg drops from
that of frog’s serum to that of pond water from the time it leaves the
ovary to the first cleavage. This decrease in osmotic pressure is pro-
bably due to absorption of water, although Backman and RunpstrOM
interpret it differently. This absorption of water, and the mixing of the
contents of the germinal vesicle with the cytoplasm, accounts for the de-
crease in viscosity of the egg after it leaves the ovary.

Literature,

Backman and Runpsrrom, 1909. Physikalisch-chemische Faktoren bei der Embry-
onalentwicklung. Biochem. Zeitschr. XXI1, 8. 390 _

_ Brataszewicz, 1908. Beitrage zur Kenntnis der Wachstumsvorginge bei Amphibien-
embryonen. Bull. de Acad, des Se. Cracovie, Math.-Nat., Okt.

Gurwitscn, 1909. Uber Primissen und anstoSgebende Faktoren der Furchung und
Zellvermehrung. Arch. Zellforschung, II, 8. 495.

Konopacka, 1908. Die Gestaltungsvorginge der in verschiedenen Entwicklungs-
stadien zentrifugierten Froschkeime. Bull. de Acad. des Se. de Cracovie,
Math.-Nat. Juli, 8. 689.

McCrenpon, 1909. Cytological and Chemical Studies of Centrifuged Frog’s Eggs. Arch.
f. Entwicklungsmech. X XVII, S. 247.

—— Chemical Studies on the Effects of Centrifugal Force on the Eggs of the Sea Urchin.

Am. Journ. Physiol., XXIII, p. 460.
On the Nucleo-albumin in the Yolk-platelets of the Frog’s Egg, with a Note on
the Black Pigment. Ibid., XXV, p. 195.
MoreGan, 1906. The Influence of a Strong Centrifugal Force on the Frog’s Egg. Arch.
f. Entwicklungsmech. XXII.
HUMBLER, 1900. Physikalische Analyse von Lebenserscheinungen der Zelle, III.
Ibid. IX, S. 63.

————

marae

Further studies on the Gametogenesis of Pandarus
sinuatus, Say.
By
J. F. Me Clendon.

(From the Zoological Laboratory of the University of Missouri and the Histo-
logical Laboratory of Cornell University Medical College, New York City.)

With 1 figure in the text and plate XVII.

In two former papers!) I considered certain phases of this subject,
but owing to the increasing interest in the part played by the chromo-
somes in heredity a more detailed study seemed advisable. The present
paper considers especially the individuality of the chromosomes during
the gametogenesis.

Material and Methods.

During the summers of 1904—6 and 1909 I collected parasitic cope-
pods at Woods Hole. The most favorable species for the study of the
gametogenesis seemed to be Orthagoriscicola muricaia KROyER®), which
occurs on the gills of the moon fish, Selene vomer, but more abundantly
on the skin of the sun fish, Mola mola. The only specimens of this species
which I was able to obtain were fixed in toto, so that a more abundant
species, Pandarus sinuatus, which occurs on the skin of the smooth dog
fish and the sand shark, had to be used. ‘The integument of the cope-
pods was partly removed or the sexual organs dissected out to insure
rapid penetration of the fixing fluid, FLemmine’s. Thin sections were
made and stained in iron haematoxylin.

————

1) Biological Bulletin, 1906, XII, p. 87 and 1907, XITI, p. 114.
2) See Proc. National. Museum, XXXII, p. 473, for description and figure.

i)

230 J. F. Me Clendon

The primary germ cell is separated at the fifth cleavage of the egg
at the ventral edge of the blastophore. It and its descendants may be
distinguished by their position in the embryo and slow rate of division.
When the germ cells have increased to four, they migrate to the defini-
tive position of the gonads and continue to multiply, but the sex cannot
forssome time be distinguished.

Spermatogenesis.

In the former study of the spermatogenesis I was chiefly interested
in tracing the development of those spermatids which do not become
spermatozoa, but are transformed into food reservoirs by the accumu-
lation of plastic products within the nuclei. In the present account the
history of the chromatin will be chiefly considered.

The spermatogonia are polyedral cells containing large nuclei the
chromatin’of which exists in the form of a reticulum associated with one
or more plasmosomes. During mitosis sixteen chromosomes may be coun-
ted, all of which are apparently equal in size.

The primary spermatocytes are smaller than the spermatogonia but
have at first a somewhat similar arrangement of chromatin, fig. 1. li
plasmosomes are present they are too small to be distinguished from
the chromatin reticulum. The chromatin reticulum soon begins to con-
dense into slender filamentous chromosomes giving the typical lepto-
tane stage, text fig. It is to be expected that some of the filaments
might be in contact when first formed, and such seems to be the case,
but I have never been able to persuade myself that they ever constitute
a continuous spireme such as has often been seen in nuclei. Hig. 2
represents an optical section showing the sixteen filaments. Erroneous
impressions may easily result from a study of optical sections, and I do
not present this as evidence of the number of filaments, which I have
determined by a careful study of whole nuclei, but such a figure gives
a better idea of the appearance of the filaments.

The filaments bunch up in the synezesis stage and at the same time
become straighter, figs. 3—6. For this reason the chromosomes of this
species are more favorable for study at this particular stage than those
of the free swimming copepods!). During the synezesis stage parallel
synapsis takes place, i. e. the chromosomes become associated in pairs
with the members of each pair parallel, figs. 3—5. The synezesis zone
occupies a considerable part of the testis. I have sectioned and studied

1) Cf. Lerat: La Cellule, 1905, XXII, p. 161.

Further studies on the Gametogenesis of Pandarus sinuatus, Say. 231

numbers of these testes, yet in no case has it been possible to count
the number of chromosomes in the early part of synezesis. The paired
arrangement of them, however, is clearly seen. Near the end of this
stage all of the filaments can sometimes be distinguished, and are eight
in number, thicker than in the leptotane stage, and double at least in’
some parts, fig. 6.
_ The eight double filaments now become dispersed toward the per-
iphery of the nucleus, giving the diplotane stage, figs. 7 and 8. The double
filaments often interlace so that they cannot be counted, although where

counting is possible eight are found. The double chromosomes now shor-

yet this might possibly be an accidental notch in the ragged outline of
the double chromosome, and further study has not cleared up this point.

Leptotane Synezesis-Synapsis Diplotane Shortening Ist. Mat. Spindle

Ata little later stage, however, a distinct second division is present, trans-
forming the double chromosome into a tetrad, fig. 10. The shape of the
tetrad is such that its longitudinal axis cannot be determined, and there-
fore it is impossible to decide whether the second division (constriction)
is longitudinal or transverse.

The two maturation mitoses then rapidly follow, distributing the
parts of each tetrad into the four spermatids, figs. 11—13. Owing to the
form of the tetrads it would be difficult to decide whether the reduction
‘in number of the chromosomes occured in the first maturation mitusis

Obviously in ordinary sexual reproduction if reduction occurs at all,
t is immaterial whether it takes place during the first or second matura-
tion mitosis. Wertsmann found that there is no second maturation divi-
i on in certain parthenogenetic eggs, and concluded that reduction is thus

oniitted. Recent investigations show, however, that if synapsis occur:

Zoo J. F. Me Clendon

reduction takes place, whether the egg be parthenogenetic or not. Shall
this fact prove universally true WrISMANN’s hypothesis is erroneous and
the question of pre- or post-reduction can have no significance in here-
dity. For this reason we will not discuss this question in relation to the
spermatogenesis of Pandarus. ‘The significant fact is that reduction has
taken place, 1. e. that each spermatid contains numerically one half of
the same individual chromosomes that were present in the primary
spermatocyte. This process is shown diagrammatically in the accom-
panying text figure.

Ocgenesis.

The oogenesis is similar to the spermatogenesis through the syne-
zesis stage. The primary oocytes are larger than the primary spermato-
cytes when first formed, and this difference increases as growth proceeds.
The leptotane stage is shown in fig. 14, and two cells in the synezesis
stage are seen in fig. 15. Immediately after synezesis a plasmosome is
found, but whether it existed during synezesis could not be determined
owing to the massed condition of the chromosomes.

Eight filaments radiate from the plasmosome and in favorable as-
pects are seen to be double, thus showing that the diplotane stage has been
reached, fig. 16. These eight double filaments arose by a longitudinal
synapsis in the synezesis stage, from the sixteen single filaments of the
leptotane stage.

The growth period now begins, fig. 16; the cell outlines first disap-
pear but reappear some time after the oviduct is reached. During the
syncytial stage the cytoplasm becomes filled with basophile granules.
Mororr!) found, in the eggs of free swimming copepods, basophile gra-
nules extruded from the nucleus into the cytoplasm, but I have not noted —
a similar origin of the granules mentioned above. The oocytes have now
become much flattened and suggest in their arrangement a pile of coins.
The yolk is deposited in the form of proteid bodies ond oil droplets. The
plasmosome becomes vacuolated and is probably in process of solution.
Fig. 17 shows a nucleus and fig. 18 a section of an entire oocyte on a
less magnified seale, at this stage. The black bodies in the cytoplasm
are proteid and the light spots are fat.

The yolk accumulates and crowds the nucleus. The nuclear sap be-
comes basophile and the nuclear wall disappears leaving the eight double
filamentous chromosomes radiating from the large plasmosome and sur-

1) Arch. f. Zellforschung, II, S. 482. ;

Further studies on the Gametogenesis of Pandarus sinuatus, Say. 233

rounded by a cloud of chromatin granules, in the centre of a yolkfree area,
fig. 19. The chromosomes have already begun to shorten. The plasmo-
some disappears, and the chromosomes continue to shorten until their
breadth equals their length, when a second, though only partial, divi-
sion transforms them into tetrads. The first division can be distinguished ’
as it is the most complete, and the tetrads become arranged on the first
maturation spindle so that the resulting mitosis separates whole chromo-
somes, and thus pre-reduction takes place, fig. 20. This reduction divi-
sion is easily followed in the related species, Orthagoriscicola muricata,
fig. 22. The second maturation mitosis is equational and separates the
halves of the resulting diads, fig. 21. A diagram of the synapsis and re-
duction is shown in the accompanying text figure, and illustrates in ge-
neral both spermatogenesis and oogenesis. The plasmosome and growth
| of the nucleus in the oogenesis are not represented.

Vs. the Critique of Hagedoorn.

| Studies relating to the individuality of the chromosomes have been
brought into special prominence owing to the debated question of the
relation of the visible parts of the germ cells to the factors discovered by
analyzing breeding experiments. | will make no attempt at a general
consideration of this subject, but wish to call attention to an argument
brought forward in a recent paper on animal breeding. HaGEpoorN?)
says: ,,[ do not see why we should attach any more value to the inheri-
tance of a chromosome than to that of any other organ or character.“
I should like to emphasize the fact that the chromosomes and the other
parts of the mitotic figure are the only ,,organs“, to use HAGEDOORN’s
term, that are seen to be transmitted bodily to the daughter cells in cell
division.
In order to depreciate the significance of the chromosomes in here-
dity, Hacepoorn states further that ,,GopLewskt — succeeded in ferti-
izing enucleated eggs of the sea-urchin with sperm of a crinoid, with the
Tesult that the ensuing larva — developed into a normal sea-urchin ga-
strula‘*. Now compare this with his further statements that ,,All inheri-
fance is Mendelian inheritance and ,,[t is impossible for a character to
€ in a recessive condition (p. 33). How can we harmonize these state-
tents? If all inheritance is Mendelian and characters of immature
es are on a par with adult characters, how does he account for the
be.
1) Arch, f. Entwicklungsmech., 1909, XXVIII, S. 2

-
e

234 J. F. Me Clendon, Further studies on the Gametogenesis of Pandarus ete.

fact that the sea-urchin © crinoid of hybrid showed only maternal cha-
racters? Certainly the crinoid is not distinguished from the sea-urchin
merely by the absence of characters. It is very doubtful whether all
inheritance is Mendelian, and GopLEWwskI’s experiment does not disprove
the hypothesis that the chromosomes bear some of the Mendelian factors.

It has not been demonstrated that the characters of the sea-urehin or
crinoid ‘larvae obey Mendelian laws, and the purely maternal character
of the hybrid might be explained on the ground that the strange sperma-
tozoon did not immediately affect the organization of the egg. TENNANT
produced hybrid echinoderm larvae showing only paternal characters
by decreasing the alkalinity of the sea water.

Explanation of Plate,

Fig. 18 was drawn with the camera lucida with Zetss apochr. homogen. ob. 2mm,

oc. 4; figs. 6—8 and 14 with oc. 12; the remainder with oc. 18 and reduced 1/3. Figs. 1
to 21 are from Pandarus sinuatus Say; fig. 22 is from Orthagoriscicola muricaia Kroyer.

Fig. 1. Spermatogonium.

Fig. 2. Primary spermatocyte, leptotane stage.

Figs. 3—-6. Primary spermatocyte, synezesis stage.

Figs. 7—8. Primary spermatocyte, diplotane stage.

Figs. 910. Primary spermatocyte, formation of tetrads.

Figs. 11—12. Primary spermatocyte, first maturation mitosis.

Fig. 13. Secondary spermatocyte, second maturation mitosis.

Fig. 14. Primary oocyte, leptotane stage.

Fig. 15. Primary oocyte, synezesis stage.

Fig. 16. Primary oocvte, diplotane stage.

Figs. 17—18. Primary oocyte, growth period.

Fig. 19. Primary oocyte, first maturation mitosis, prophase.

Fig. 20. Primary oocyte, first maturation mitosis.

Fig. 21. Secondary oocyte, second maturation mitosis.

Fig. 22. Primary oocyte, first maturation mitosis, side view of equatorial plate.

APChiV ff. Lellrorschung DC.?. Ps LQ. AYL.

~ * . oe = — ——
| ——y (Teens eee SS ae Ss —

| [Reecenttesneeeeeeererree ee — ¥
| we Clend == — == = - >

} Me Clenidon & Kline del Verlag xv Wilhela Engelmanr
|

|

|
:

On the Dynamics of Cell Division.

I. The Electric Charge on Colloids in Living Ceils in the Root Tips
of Plants.

By
J. F. McClendon.

(From the Histological Laboratory of Cornell University Medical
College, New York City, U.S. A.)

With 2 figures in text and plate III.

Eingegangen am 26. April 1910.

The electric charges carried by ions have been extensively in-
vestigated and have yielded such valuable data that a similar study
of colloids seems advisable. Here however we meet with difficulties,
for many colloids are precipitated in the absence of electrolytes and
the electrolytes may determine the sign of the charge on the colloids.
Thus egg albumin is positive in acid and negative in alkaline solution.

Many complex colloids are positive, whereas pepsin is negative’).
Nuclei usually go toward the anode and cytoplasm to the cathode’).

Last summer I began experiments on the cataphoresis of colloids
in the living cell in order to test certain theories as to the cause of
movement of granules and chromosomes accompanying cell division.
These experiments are still in progress. I found that the nucleus
as a whole did not bear the negative charge, which was localized
in the chromatin, whether the latter was in the form of granules,
Spireme or chromosomes. The chromatin when carried toward the
anode by as strong a current as was tried (in the case of Dremyctilus

1) Journ. Exp. Med. IX. p. 86 and 254. Biochem. Zeitschr. XXIV. S. 53.
?) R. Linu, Am. Journ. Physiol. VII. p. 273. Henri, Compt. rend. Soc.
de Biol. LVI. p. 867.

On the Dynamics of Cell Division. I. 81

not exceeding .02 amperes to the square mm. for 30 min.) was re-
tained by the nuclear wall when the latter was present. ‘The mitotic
figure was sometimes moved as a whole toward the anode, and the
chromosomes were never pulled out of it or carried through the
cell wall.

ia) zane

ays

We
vey

=) Te
= bor mb}

A portion of a longitudinal section ofa hyacinth root through which an electric current of .0005 ampere

was passed for 30 minutes, stained with safranin. In the figure the anode was below and cathode above.

The basophile substances were carried by the current toward the anode. In sectioning the microtome

knife passed in the direction of the figure from right to left, and some of the chromosomes were torn
out of the spindles by the microtome knife, and carried toward the left.

I found that the nucleoli, pigment granules and yolk platelets
of frog’s egg also traveled toward the anode.

In the meantime the paper by PentiaLui on the cataphoresis
of the chromosomes in the root tips of the hyacinth appeared‘). He
States that the chromatin is more and more affected by the current

1) Influenza della corrente elettrica sulla dinamica del processo cariocine-
tico. Arch. f. Entw.-Mech. XXVIII. 8S. 210.
Archiy f. Entwicklungsmechanik. XXXI. 6

82 J. F. McClendon

as the process of mitosis progresses, ‘and that with a current of
about .00005 amperes lasting 30 minutes some of the chromosomes
are torn away from the spindle and carried through the cell wall

Fig. 2.

Photograph of another portion of the preparation shown in text fig. 1 and with the same orientation.
The mitotic figures are not distinct, but the accumulation of basophile substance in the anodal ends
of the cells and nuclei may be observed. >

toward the anode. Such results are interesting indeed, and if veri-
fied would tend to lend support to dynamic theories of the nature
of the mitotie figure. But it might be considered unusual to find
chromosomes being carried through cellulose cell walls by such weak

On the Dynamies of Cell Division. I. 83

currents. The experiments appeared to be of sufficient importance
to warrant repetition. After an extensive series of experiments I
failed to convince myself that PenTIMALLI’s interpretation of his re-
sults was correct. In the controls many chromosomes were displaced
by the microtome knife, even when the sharpest and smoothest blade
was used. The fact that chromosomes were not carried by the
electric current through the cell walls of animal cells, in which dis-
placement of chromosomes by the microtome knife seldom occurs,
suggested that the chromosomes which PENTIMALLI supposed were
moved by the current were in reality moved by the knife in cutting
the sections. This suggestion was supported by the fact that with
an extensive graded series of currents including those used by
PENTIMALLI, many of the mitotic figures showed no displacement
of chromosomes, whereas with those currents which moved the chro-
matin in the resting nuclei, all of the nuclei in the root tip showed
this phenomenon, text figs. 1 and 2. Prnrmanir allowed me to
examine one of his preparations, for which my sincere thanks are
due him. It did not convince me that my interpretation of my re-
sults was erroneous. Since these experiments may so easily be
repeated on onion as well as on hyacinth roots and should be in-
cluded in every course in Cytology, the reader may have a chance
to interpret the results for himself.

PENTIMALLIs conclusion that the chromatin bore a negative
charge was confirmed by my experiments, which did not however
support his conclusion that the cataphoresis increases as the process
of mitosis advances. The chromosomes seem to be firmly attached
to the spindle so that in order to move them by the current the
whole spindle must be moved, a process which, as will be shown
later on, requires more current than to move the chromatin granules
in the resting nucleus. The charge on the chromatin may increase
as the process of mitosis advances as PENTIMALLI supposes, but the
resistance offered by the spindle prevents proving such a supposition
by this means.

Material and Methods.

Hyacinth and onion bulbs were set in damp sawdust or in tap
water until the roots were of sufficient length to be handled. In
the onion, more mitosis are found at about 1 p.m.') than at any

1) Kexuicorr, Bull. Torrey Bot. Club. XXXII. p. 529.

6*

84 J. F. McClendon

other hour of the day, but this difference is so slight that it was
hardly worth while to delay experiments until that hour. In roots
grown in tap water sometimes very few mitoses were to be found,
but a very large number of mitoses is a disadvantage and the con-
venience of this method made it useful.

Various forms of electrodes were tried. One form that may
commend itself to those who wish to repeat the experiments without
much trouble can be easily set up, provided a direct current of about
110 volts is available, as follows:

One wire of a lamp cord is cut and the cotton insulation re-
moved from 2 inches or more of the cut ends. The rubber insulation
is removed from an inch or more of the cut ends, exposing the
copper wires, which are then introduced into glass tubes of such
size that they may be tightly plugged by the rubber insulation.
These tubes, while being adjusted, are completely immersed in copper
sulphate solution, and on removal from the solution their free ends
are plugged with absorbent cotton soaked in tap water, and fitted
to rubber tubes filled with tap water or salt solution. The free ends
of the rubber tubes are then plugged with absorbent cotton soaked
in the same fluid with which they are filled, and the electrodes are
ready to be applied to the tissue, provided no air bubbles have
gotten into the tubes.

As the current is determined by the sum of resistances of the
lamp, of the fluids in the glass and rubber tubes and of the tissue,
it may be varied by varying any of these resistances. The resistance
of the copper sulphate solution and of the tissue may be made
negligable. To change the current it is most convenient to change
the resistance of the lamp or of the fluid in the rubber tubes. This
is done by substituting a lamp of a different candle power or
changing the length or the salt content of the rubber tubes.

PENTIMALLI used physiological salt solution to fill the rubber
tubes. The roots are not normally exposed to sodium chloride of
such high concentration, and it would seem safer to fill the rubber
tubes with tap water, to which may be added a very little salt if
necessary.

If the rubber tubes are of sufficient length there will be no
danger of the copper reaching the tissue, otherwise some proteid
should be added to the fluid in the rubber tubes to precipitate the
copper, or the more complicated electrodes described by PENTIMALLI
substituted.

eee -.hC tS

On the Dynamics of Cell Division. I. 85

The current was measured with a galvanometer!) which was
sensitive enough for all except the greatest densities of current used,
but was not calibrated with very great accuracy. The cross section of
the current in the tissue was made approximately constant by using
root tips of about the same size and running the current longitudinally,
except in a few experiments in which the current was run crosswise.

After the passage of the current, the root tip was fixed in
Boury’s fluid for 30 minutes, sectioned serially and stained in safranin,
sometimes followed by methyl blue (wasserblau). Usually the latter
was dropped on one end of the slide so that some of the sections
had the safranin only while others had the double stain. Care was
taken to record the direction of the current and also to have it pass
in the plane of the section.

Experiments.

As PENTIMALLI used the hyacinth and as the cells in its roots
are about twice the diameter of those in the onion only the results
on the hyacinth will be described. The results on the onion present
no material difference.

The amount of cataphoresis would depend on the density and
duration of the current and could be changed by varying either, but
as a change in duration of the experiment might involve a change
in the number of phases during which a single cell was subjected
to the current, it was thought best to vary the current density and
have the time factor constant. In most of the experiments the current
was passed for 30 minutes and varied from .QQO01 to .01 amperes.
PENTIMALLI used about .00002 to .00005 amperes for 20—45 minutes.

I first thought it necessary to determine whether the current
killed the cell. To do this currents were passed through roots still
attached to bulbs placed in a moist chamber, the roots being marked
by colored threads. In case the cells of the root tip were killed,
the root ceased growing after one or two days and the tip rotted,
but when the proximal part of the root was killed, the whole root
rotted. It was found necessary to section roots some time after the
passage of the current to determine more exactly the extent of the
necrosis. Whether these lethal effects of strong currents were due
to the migration of ions or of colloids, I have no means of deter-
mining. The general result was that if the current was much greater

1) For the use of which I am indebted to Dr. MAx Morsr.

86 J. F. MeClendon

than the minimal amount necessary to produce visible cataphoresis
of the colloids, some of the cells disintegrated within a few days.
I think it safe to assume however that all of the cells maintained
many of the properties of living matter during the passage of the
current except perhaps with the maximum current used. PENTIMALLI
states that in using currrents of .004 to .005 amperes, the resistance
of the tissue fluctuated and finally increased so that the current
ceased altogether, indicating rupture of cell walls and disintegration
and death of the cell. Such changes may indicate death of the cell
but I know of no reason for supposing this to be the case, unless
the final high resistance be taken to indicate coagulation of the
proteids. The resistance of many tissues decreases during stimulation —
and shows a permanent decrease after death. Root tips sectioned
after the passage of the strongest currents which I used, showed
considerable plasmolysis of the cells but no rupture of membranes.

In each of the first seven experiments a control root was cut
in serial sections. A resting cell is shown in fig. 1, plate UI. It will
be noted that both cytoplasm and nuclear contents are almost uni-
formly distributed. The plasmosome shown is excentric, but as in
most cells there were two plasmosomes present, one of which does
not appear in the optical section. The chromatin is granular and
no definite reticulum is observed.

A special search was made for abnormalities, as these might
aid in the interpretation of the experiments. Sometimes the plasmo-
some was dragged out of the nucleus by the microtome knife. More
often however the mitotic figures were affected, the microtome knife
displacing chromosomes or portions of the spireme, figs. 6—11. In
some cases the spindles were diagonal, fig. 9, an unusual but probably
not an abnormal occurrence. In a few cases the axis of the spindle
was curved, fig. 7.

With the minimum current used no effect is observed. As the
current is increased to about .00005 or .0O01 amperes, the basophile
substances, shown by darker shading in the plate, are carried toward
the anode, fig. 2. This effect is more evident in the nucleus, which
contains large amounts of basophile substance, the chromatin, but is
also distinct in the cytoplasm. As the current is increased to .00015
amperes or more, the basophile substances are still further concen-
trated in the anode ends of cells and nuclei, and only acidophile
substances remain in the cathode ends. Whether the acidophile sub-
stances are moved by the current cannot well be determined, as

On the Dynamics of Cell Division. I. 87

their distribution in the controls is obscured by the intermingling of
pasophile substances. With a current of about .01 amperes the
nuclear membrane is pushed out towards the anode by the pressure
of the chromatin, fig. 4, but apparently the cathodal end of the
nuclear wall was moved very little if at all. Whether the nucleus
changed in volume during the process, was not determined.

It might be objected that this alteration of the appearance of
the stained section is not due to the movement of basophile or acido-
phile substances, but is accounted for by the alteration of the affini-
ties of the different parts for stains, due to the accumulation of
acid at the anode and alkali at the cathode, forming acid and alkali
albuminates. Such an accumulation of acid and alkali would neces-
sarily take place if the surface layers of cells and nuclei offered
more resistance to the passage of ions than the interior, and there
is much evidence to indicate that such is the case for cells in general.
But the tissue was fixed in a strongly acid fluid, and we should
then expect all of the protoplasm to be basophile. Furthermore, in
the prophase of mitoses the spireme is carried toward the anode,
fig. 5, demonstrating undoubtedly the cataphoresis of colloids, which
are in this case perhaps in the »gel« phase. We may assume then
that acid appears at the anode and alkali at the cathodes of both
cell and nucleus, but this accumulation does not account for the re-
distribution of basophile and acidophile substances, which is due to
the migration of colloids.

As the process of mitosis advances the chromatin is less and
less affected by the current. Whereas .0015 amperes are sufficient
to move the spireme, fig. 5, .005 amperes are required to move the
spindle, fig. 16, which is often hardly affected by 0.1 amperes, fig. 12.
However in the cell shown in fig. 12 the movement of the spindle
may have been stopped by the cell wall. The cytoplasm is very little
affected by the current during mitosis. This may be due to the re-
sistance offered by the mitotic figure to the movement of colloids.

When the current passes during the later phases of mitosis the
cell division is displaced toward the anode, fig. 17. This is a demon-
stration that cataphoresis takes place during the life of the cell, for
cell division is a vital process, and the formation of a new cell wall
in this case evidently took place after the displacement of the mitotic
figure by the current.

The study of the effects of the current on the mitotic figure is
complicated by the displacement of the chromosomes by the micro-

88 J. F. MeClendon

tome knife in cutting the sections. In controls a considerable amount
of displacement occurs, and in regions with relatively few mitoses
it is easily observed that the displacement always occurs in the di-
rection of the movement of the knife. Where mitoses are numerous
this is not so evident, owing to the fact that chromosomes of one
mitosis‘ may be carried to the vicinity of another mitosis. After
passage of the current the same appearances were observed. By
picking out individual mitoses in certain slides it is very easy to
find numerous instances where the chromosomes are displaced toward
the anode, even through cellulose cell walls. But by careful study
of many whole series of sections very different observations may be
made, especially if the direction of the microtome knife was purpo--
sely chosen so as to be at right angles to the direction of the current,
as it was in the large majority of my experiments. In text figure 1,
it may be noted that the direction of the current as marked by the
polarization of cells and nuclei, is toward the top of the page,
whereas the displacement of chromosomes is toward the left. On
the other hand, after the passage of a current of any density within
the limits above mentioned, many spindles showing no displacement
of chromosomes were found, figs. 12—15.,

Conclusions.

PENTIMALLI’s assertion that the effects of the passage of an
electric current through the cell demonstrates that the chromatin
bears a negative electric charge is confirmed by the experiments
described above. If the chromosomes were attracted to the asters
or poles of the spindle because the poles are positive, the chromo-
somes being negative, we would expect to find the yolk platelets
and pigment granules of the frog’s egg also attracted, as they bear
negative charges. The pigment granules in the frog’s egg are con-
sidered by some observers to be attracted toward the aster, though
a considerable area around the centrosome is always free from them.
However I think that everyone will agree that the yolk platelets
are repelled by the aster. The fact that the negative chromosomes
are attracted, and the negative yolk platelets are repelled by the
aster shows that the mitotic figure is not simply a bipolar electric
field. Furthermore if the poles of the spindle bear positive charges
and the chromosomes negative, we should expect the chromosomes
to move toward the anode and the poles of the spindle to move

-

On the Dynamics of Cell Division. I. 89

toward the cathode on the passage ofa current, which was not found
to be the case. We should also expect the spindle in the meta- or
anaphase to break in two at the equator when the cell contents are
subjected to violent mechanical disturbance since there is no attrac-
tion between the two halves of each divided chromosome, but on
the contrary the mitotic figure resists mechanical distortion to a re-
markable degree.

The forces concerned in mitosis may be electric, but in ultimate
analysis all forces may finally be reduced to electric components.
To suppose that the spindle consists of proteids in the »gel« phase
and that it »grows<, does not explain the phenomenen. We know that
osmotic forces enter largely into the growth of plants, but osmotic
membranes in the mitotic figure would be difficult if not impossible
to demonstrate. The reduction of mitosis to a readjustment of surface
tension in the walls of demonstrable or hypothetical alveoli, as has
been done by RuumBLER, following the work of BirscHLi, suggests
itself as an alternative, and is worthy of further investigation.

The appearance of cells through which electric currents have
been passed is similar to that of those which have been centrifuged.
In many cases investigated, the substances of greater specific gray-
ity bear the negative charge, and thus the effects of centrifugal
force and the electric current appear to be the same, but this is
probably a mere coincidence. Whereas the bodies moved by the
centrifuge are largely stored materials, the electric current has been
shown to move ions, enzymes and yarious thermo-labile bodies,
which may or may not be in solution. In other words, we have in
the electric current a little used method of investigating life pro-
cesses which might possibly be applied as centrifugal force has
been. In much of the work with the electric current the end results
have been recorded but the intermediate steps not observed. The
separation of basophile from acidophile protoplasm by the electric
current points out a method of observing the immediate effects of
the electric current on tissues. It would be interesting to trace the
relation between these immediate efiects and the abnormal mitoses
which have been observed in tissue subjected some time previously to
the electric current.

Summary.

In the cells of root tips of the onion and hyacinth the basophile
Substances migrate toward the anode on the passage of an electric

90 J. F. MeClendon, On the Dynamics of Cell Division. I.

current, except in case of the mitotic figure, which migrates as a
whole toward the anode.

As the process of mitosis advances the effect of the current on
the chromatin decreases, contrary to the conclusion of PENTIMALLI,
who supposed that it increases.

Chromosomes or other bodies are never carried through the nuclear
or cell walls by currents of the density used (.00001—.01 amperes).

Zusammenfassung,

In den Wurzelspitzen der Zwiebel und der Hyazinthe wandern die baso-
philen Substanzen beim Durchgang eines elektrischen Stromes nach der Anode
hin, auBer wenn eine mitotische Figur vorhanden ist, welche im ganzen nach -
der Anode hinwandert.

Mit dem Vorschreiten des mitotischen Prozesses nimmt die Einwirkung des
Stromes auf das Chromatin ab, entgegen der Folgerung von PENTIMALLI, welcher
annahm, da®B sie wiichst.

Chromosomen oder andre Kiérper werden durch Stréme von der verwen-
deten Stiirke (0,00001—0,01 Ampére) niemals durch die Kern- oder Zellwiinde

hindurchgetrieben. (Ubersetzt von W. Gebhardt.)

Explanation of Plate Ill,

All the figures were drawn with a camera lucida, with B. & L. homogen.
imm. obj. 2mm., oc. 1. All are from longitudinal sections of root tips of the
hyacinth. The figures are so arranged that the microtome knife passed from
right to left in cutting the sections, and in case a current was passed, the anode
was below and cathode above, and the duration of the current was 30 minutes.
The basophile substances are distinguished by darker shading.

Fig. 1. Control.

Fig. 2. Subjected to a current of .0001 amperes.
Fig. 3. - - - - - 00015 -
Fig. 4. - - - - - O1 -
Fig. 5. - - - - - .0015 -
Fig. 6. Control.

Fig. 7. -

Fig. 8. -

Fig. 9. -

Fig. 10. -

Fig. 11. -

Fig. 12. Subjected to a current of .01 amperes.
Fig. 13. - - - - - 0015 -
Fig. 14. . - - : - .0002 -
Fig. 15. - - - - - .0006 -
Fig. 16. - - - - - .003 -
Fig. 17. : <) Etat Oe gee OI :

Archiv fir Entwicklungsmechanik Bd. XXX1 Taf I 90°

Verlag von Wilhelm Engelmann x Leivzig Lith. Anst. Johannes Arndt, Jena

Reprinted from the American Journal of Physiology.
Vol. XXVII. — December 1, 1910.— No. II.”

ON THE DYNAMICS OF CELL DIVISION. —II. CHANGES
IN PERMEABILITY OF DEVELOPING EGGS TO ELEC-
TROLYTES.

By J. F. McCLENDON.

[From the Histological Laboratory of Cornell University Medical College, New York City,
and the Laboratories of the Carnegie Institution at Tortugas, Fla., and the U. S. Bureau
of Fisheries at Woods Hole, Mass.]

HE process of cell division may be divided into two distinct phe-
nomena, the division of the nucleus and of the cytoplasm. Al-
though these processes are closely interrelated, they can occur sepa-
rately. Karyokineses may occur without subsequent cytoplasmic
division, and cytoplasmic division may occur without the presence of
a nucleus or chromatin in any form.! The division of the cytoplasm
in plant cells is accomplished by the formation of a division wall, but
in most animal cells by simple constriction.

The constriction of the cell seems to be a special case of these pro-
toplasmic movements that were shown by Quincke to resemble move-
ments accompanying surface tension changes.” If the constriction of
the cytoplasm were due to surface tension changes, we should expect
a band of greater surface tension to include the cleavage furrow. In
this case there would be a flowing of the superficial cytoplasm from
the poles of the cell toward the cleavage furrow, and of the deeper
protoplasm in the opposite direction.

By a study of fixed material Nussbaum * showed movements of the
pigment granules in cells of frog’s embryos from the interior to the sur-
face and along the surface to the position of the future cleavage furrow.
On constriction of the cell the granules were massed in the form of a
plate in the cleavage plane. These movements indicate the surface
tension changes described above.

1 McCLenpDon: Archiv fiir Entwicklungsmechanik, 1908, xxvi, p. 662.
2 Buetscuui: Archiv fiir Entwicklungsmechanik, 1900, x, p. 52.
3 NussBAum: Anatomische Anzeiger, 1893, vill, p. 666.

240

On the Dynamics of Cell Division. 241

Conklin * found evidence for such movements in the changes in posi-
tion of certain structures in Crepidula eggs.

The first observation of this process in living cells was made by
Erlanger,? who saw movements of superficial granules toward the
cleavage furrow, and of internal granules toward the poles, of Nema-
tode eggs.

Gardiner ® observed, in living eggs of Polychcerus caudatus, colored
granules move to the surface and then along it to the position in which
the cleavage furrow appeared immediately afterward. Fischel’s ob-
servations are considered below.

The fact that cells usually round up before cleavage, if not previ-
ously spherical, indicates a general increase in surface tension, and it
is only necessary to assume a greater increase to be localized along the
cleavage furrow to account for the constriction.

Robertson ‘ floated an olive oil drop on water and laid across it a
thread moistened with soap (or soap-forming) solution. After the
thread reached the edges of the drop the latter was torn in two. Since
soap decreases the surface tension between oil and water, he concluded
that cytoplasmic division is due to a decrease in surface tension along
the cleavage furrow. As this view has been accepted by Lillie * and
Loeb,’ it seems worth whlie to point out Robertson’s error.!° In Rob-
ertson’s experiment three different surface tension films occur, between
air (A) and water (W), air and oil (O), and water and oil (Fig. 1), and
an equilibrium is established when the water-air surface tension equals
the horizontal components of the air-oil plus the oil-water surface
tensions. When the moistened thread is laid across the oil drop, two
nore films are added, 7. e., air-soap solution (.S) and oil-soap solution
(Fig. 2). At opposite edges of the drop where the thread touches the
water, the soap would decrease the water-air surface tension, and the
undiminished pull on the remainder of the edge of the drop would pull
it in two (Fig. 3).

4 CoNKLIN: Biological lectures at Woods Hole, 1908, p. 69.

5 ERLANGER: Biologische Centralblatt, 1897, xvil, p. 152.

6 GARDINER: Journal of morphology, 1897, x1, p. 55.

7 Ropertson: Archiv fiir Entwicklungsmechanik, 1909, xxvii, p. 29.
Littte: Biological bulletin, rg09, xvii, p. 203, footnote.

° Lorp: Chemische Entwicklungserregung des tierischen Eies, p. 5.
© This was first pointed out by me before the American Society of Zodlogists,

see Science, xxxi, p. 467. It was discussed by A. B. MAcALLuM, Science, 1910,
XXXli, pp. 498-500.

8

ee ee eer ne

242 J. F. McClendon.

I have repeated Robertson’s experiment and also modified it by
entirely submerging the oil drop. Enough alcohol was added to the
water to make the oil sink below the surface, and the soap sqlution
introduced through a capillary pipette, or a piece of solid soap held
near the oil drop, or a thread covered with solid soap was wrapped
around the oil drop. Very little movement of the oil occurred, but

FicurEs 1 to 3.— A, air; O, oil; W, water; S, soap solution. The arrows show the
direction of the pull of surface tension. The dotted line in Fig. 3 bounds the soap
solution. Further explanation in text.

that which did occur was always a bulging toward the soap, and never
a constriction or receding from the soap. Similar experiments were
also tried on the under side of oil drops floating on water, and unless the
soap reached the water-air film, the oil advanced toward the soap and
no constriction occurred. We may conclude, then, that the cleavage
furrow is a region of increased surface tension, as shown by Biitschli
and others, and not of decreased surface tension, as Robertson, Lillie,
and Loeb maintain.

One may ask why such movements as seen by Erlanger, Gardiner,
and others have not been observed in all dividing cells that have been
studied alive. The answer to this may be sought in the structure or
consistence of protoplasm. If the cytoplasm present an alveolar
structure, the spreading of the surface in regions of reduced surface
tension would be almost entirely confined to the individual alveoles,
and the general effect would be a slow stretching of the surface in

On the Dynamics of Cell Division. 243

areas of less surface tension and a slow contraction of the surface in
areas of greater surface tension, as shown by Biitschli’s microscopic
oil foams. The constriction of the cleavage furrow would then take
place as though a rubber band around the cell contracted.

In the constriction of the egg of the sea urchin, Arbacia punctulata,
usually just such a number of chromatophores are carried into the
cleavage furrow that when the two daughter cells are formed the pig-
ment is evenly distributed over all parts of their surfaces. Under cer-
tain abnormal conditions in which the cleavage is more violent the
pigment is massed in the furrow. Fischel observed a massing of pig-
ment along the lines of the future cleavage furrows in the eggs of Ar-
bacia pustulosa. The reasons we get so little movement of pigment in
the egg of Arbacia punctulata probably are the alveolar structure and
the presence during cleavage of the “hyaline plasma layer,” or ‘‘ Ver-
bindungsschicht,”’ to which the surface movements are chiefly confined.
The hyaline plasma layer is said to be formed by a recession of granules
toward the interior of the egg, leaving the superficial layer free from
granules and almost invisible. It is formed before the first cleavage
and becomes heaped up in the cleavage furrow. Whenever such an
outer layer occurs, as in eggs of sea urchins and ctenophores, it be-
comes heaped up in the cleavage furrow; this indicates increased sur-
face tension in this region (or decreased surface tension at the poles).

In the cutting off of the micromeres in the Arbacia egg the pigment
entirely disappears from the micromere pole, indicating spreading
movements due to the surface tension being less here than in the re-
gion of the future cleavage furrow. Similar movements of granules
have been observed in the cutting off of polar bodies in various eggs,
and it may be concluded that for the separation of a very small cell
from a large mass of protoplasm a very great difference in surface ten-
sion between the pole of the small cell and the cleavage furrow is
required.

Changes in surface tension may be the result of the presence of cer-
tain substances in one of the two fluids in contact, changes in tempera-
ture, or a difference in electric potential across the boundary. In
numerous instances electric changes have been found to accompany
vital movements. Hyde” detected electric changes accompanying

11 GotpscHmipt and Poporr: Biologische Centralblatt, 1908, xxviii, p. 210.
12 Hype: This journal, 1904, xii, p. 241.

244 Seok. McClendon.

cleavage, and the question whether the constriction of the cytoplasm
is due to electric changes seems worth investigation.

If an electric current were passed through a solution containing a
living cell, and if the cell surface offered more resistance to the passage
of ions than either the medium or the cell interior, a difference of po-
tential would be produced between the inner and outer sides of the
cell surface, and would be proportional to the angle that the surface
made with the current lines cutting it, z. e., it would be greatest at the
point where the surface was at right angles to the current lines and
equal to zero at the point where the surface was parallel to the current
lines. The surface tension would be reduced at the poles (the points
nearest the electrodes), and the equator would lie in a region of rela-
tively greater surface tension. This would result in the protrusion of
the polar regions and constriction of the equator, thus producing the
form change of the first stage of cleavage.

When a current of a certain density was passed through an unfer-
tilized Arbacia egg, the surface nearest the anode showed spreading
movements and bulged out. We might conclude from this that this
egg was less permeable to anions than to cations. The confined anions
caused a difference of potential between the two sides of the surface
nearest the anode, thus decreasing the surface tension, and spreading
of the surface and bulging of the egg followed. At the surface nearest
the cathode, the anions that could not enter could pass around the
egg, and therefore the difference of potential was not so great as at the
opposite pole. If the egg were as poorly permeable to cations, we
should expect a reduction of surface tension at the pole nearest the
cathode.

It seemed to me that an analysis of artificial parthenogenesis might
throw light on the question of cell division, and in the summer of 1909
I began an attempt in this direction.

ARTIFICIAL PARTHENOGENESIS.

It is well known that the eggs of different individuals of the same
species vary in response to stimuli. Investigators usually suppose that
they have normal material when a large per cent of the eggs develop
in a control to which sperm is added. In order to test this method of

On the Dynamics of Cell Division. 245

controlling experiments and be sure no unknown factor vitiated the
results, I made a series of experiments by fertilizing eggs of the same
mother with sperm of different fathers, and eggs of different mothers
with sperm of the same father, with the following results, giving the
percentage of eggs developing:

Mothers.
Fathers.

Cc

93
94

92

90

It may be seen by inspecting the table that development depends more
on the eggs than on the sperm, so the practice of keeping a fertilized
control seems to be a good one. Fertilized and unfertilized controls
were kept to all of my experiments, and the experiment thrown out if
fertilization did not occur.

The question which concerns us first is, in what ways have cells
been caused to divide, and maturation (in most cases), as well as seg-
mentation, is cell division. We may summarize the methods used as
follows:

Hypotonic solutions (distilled water, Schiicking).

Nearly isotonic solutions made by adding to sea water or to distilled
water the following substances:

Acids (Delage, Fischer, Herbst, Lefevre, Loeb, Lyon, Neilson,
Schiicking, Tennent).

Alkalis (Delage, Loeb, Schiicking).

Neutral salis (Delage, Lillie).

Hypertonic solutions:

Acids (Delage, Loeb).

Alkalis (Delage, Loeb).

Neutral salts (Bataillon, Bullot, Delage, Fischer, Hunter, Kosta-
necke, Loeb, Lyon, Mead, Scott, Treadwell, Wilson).

246 J. F. McClendon.

Non-electrolytes (Delage, Loeb).

Mechanical shock (Delage, Fischer, Mathews, Scott).

Thermal changes (Bataillon, Delage, Greeley, Lillie, Loeb, Schiicking).

Electric changes (Delage, Schiicking).

KCN or lack of oxygen (Loeb, Lyon, Mathews).

Fat solvents (Loeb, Mathews).

Alkaloids and glucosides (Hertwig, Loeb, Schiicking, Wassilieff).

Blood sera (Bataillon, Loeb).

Soap and bile salts were found effective by Loeb when followed by
hypertonic solutions.

As all of these agents were not tried and found effective on eggs of
the same species, their differences might be thought to correspond to
differences in the eggs, and therefore I thought it worth while to try a
large number of them on the egg of Arbacia punctulata at Woods
Hole, Mass. Segmentation was produced by the following methods:
the time indicated is the optimum duration found after a series of ex-
periments, the tables being omitted:

Hypotonic solutions (70 c.c. sea water + 30 c.c. tap water or dis-
tilled water, one to one and a half hours).

Nearly isotonic solutions:

Acids (50 c.c. sea water and 3 c.c. 1/10 normal acetic, fifteen to sixty
seconds. Or sea water charged with CO, in a “‘sparklet fountain,”’
five to ten minutes).

Alkalis (1.2 c.c. 1/10 normal NH,OH or NaOH + 50 c.c. sea
water, twenty to sixty minutes).

Salts (5/8 normal NaCl, one-half to two hours, this is very slightly
hypertonic).

Hypertonic solutions (100 c.c. sea water + 15 c.c. 214 normal NaCl,
one hour. Or sea water boiled down to .76 of its volume, one hour.)

The eggs were also made to segment by placing them in sea water
brought from Boca Grande Key, twelve miles west of Key West,
Florida, in steamed out, glass-stoppered and paraffine-sealed bottles.
The eggs were allowed to lie twenty minutes in Woods Hole sea water,
then placed for two and a half hours or four hours in Boca Grande sea
water and returned to Woods Hole sea water, or allowed to remain in-
definitely in Boca Grande sea water. At the end of nine hours seg-
mentation had occurred in all three lots, about ro per cent were seg-
mented in that left four hours in Boca Grande sea water.

On the Dynamics of Cell Division. 247

Woods Hole sea water has a A = 1.818, specific gravity = 1.024
(Garrey). Boca Grande sea water has a A = 2.05, specific gravity =
1.0248. The alkalescence of the Boca Grande sea water is greater
than that of Woods Hole. The hypertonicity and greater alkalinity
may have both aided in producing the segmentation. This experi-
ment producing such poor results (10 per cent), is given so much
space merely because the effects were produced by natural sea water.
However, this does not prove that Arbacia grown at Boca Grande
would be naturally parthenogenetic.

Mechanical shock (shaking in vial, by hand, five minutes. Or pour-
ing from one dish to another every ten minutes for three hours. One
effect of agitation is the removal of the jelly-like covering from the
eggs, after which, perhaps, the mere contact of the egg with another
surface will start development. Mathews supposes the effect of shak-
ing is rupture of the nuclear wall, at least in the starfish egg).

Thermal changes (keeping at 32° C., four minutes; keeping at 1° C.,
one to eleven hours; or at 10° C., one to twenty hotrrs. By the end of
twenty hours some were already segmented).

Electric changes (several entire ovaries were placed in longitudinal
series in a glass tube, and an alternating current from a small induc-
tion coil was passed through it for two hours. To avoid error from
polarization at the electrodes, only the eggs from the central ovary
were observed).

KCN or lack of oxygen (a stream of hydrogen eight to twenty-two
hours, with or without previous boiling of the sea water. Or 1/500 nor-
mal KCN, seventeen to thirty-two hours. Or 1/1000 normal KCN
thirty-two hours).

Fat solvents (%4 saturated solution of ether in sea water, ten
minutes).

Certain combinations such as carbonic or fatty acid followed by
hypertonic sea water, or tannic acid + an excess of NH,OH,* or tannic

13 In making his ‘“‘ammonium tannate” solution, DELAGE considered tannin a
hexivalent acid, but I can find no confirmation of this view in chemical handbooks.
On adding this solution to sea water, a slight precipitate, probably calcium or
magnesium tannate, forms, and the fact that DELAGE obtained as good results
by adding the ‘‘ammonium tannate” to a sugar solution does not prove that the
precipitation of some salt in the sea water solution was not in this case a factor in
the production of parthenogenesis.

248 J. F. McClendon.

or acetic acid followed by NH,OH or NaOH, seemed to produce better
results than the single treatments.

The following results were obtained on other species:

First, at Woods Hole, eggs of Cumingea and Mytilus were caused
to maturate by treatment with hyperalkaline sea water.

Second, at Tortugas, Fla., where I found the A = 2.03, specific
gravity = 1.0246, and alkalescence greater than at Woods Hole.

A few of the immature eggs of Ophiocoma rizii maturated when
left twelve hours in 50 c.c. sea water + one drop of dilute ammonia,
whereas none maturated in the control.

Eggs of Toxopneustes (Lytechinus) variegatus and Tripneustes
(Hipponée) esculentus were made to segment by placing a test
tube of sea water containing them for one minute in water at
38°-44° C., then pouring the eggs into a dish of sea water at the
normal temperature.

After treatment with sea water carbonated in a “‘sparklet syphon,”
followed by hypertonic sea water, development went farther in both
species than after any of the single treatments tried. By increasing
the duration of the treatment the rate of development was increased
(approached or equalled that of fertilized eggs), but the percentage of
resulting larve decreased, indicating injury to the eggs.

The optimum for Toxopneustes was: carbonated sea water one
and one-half to five minutes, followed by 100 c.c. sea water + 16 C.c.
2% normal NaCl, thirty to forty-five minutes. If eggs had remained
a long time in sea water before the beginning of the experiment, a
shorter stay in carbonated sea water was required than if they had
been just taken from the ovaries. In this connection it is to be re-
marked that carbonated sea water hastens the solution of the jelly-
like coverings, which takes place more slowly in natural sea water.

The optimum for Tripneustes was carbonated sea water ten minutes,
followed by hypertonic sea water one hour. If we look for something in
common in all of these methods of artificial parthenogenesis, we meet
with many difficulties. I concluded that all of the methods of artificial
parthenogenesis could not directly initiate any one single chemical
reaction in the egg, but must have their first common effect in some
physical or physico-chemical change.

The osmotic methods have this in common, that in all there is an
increase in osmotic pressure. If the eggs are placed in sufficiently

On the Dynamics of Cell Division. 249

concentrated sea water or other hypertonic solution, some may seg-
ment while remaining in the hypertonic solution, but if placed in dis-
tilled water (Schiicking) or diluted sea water (McClendon) they
segment only after removal to natural sea water, which means an
increase in osmotic pressure of the medium. I do not, however, con-
clude from this that in the latter instance it is the return to sea water
rather than the sojourn in the hypertonic solution that starts the de-
velopment of the egg.

Traube “ showed that ‘fertilization membrane-forming”’ substances
are effective in greater dilution the more they lower the surface tension
of water. If the egg surface contain lipoids, such substances will be
adsorbed or absorbed by the lipoids in the ratio that they lower the
surface tension of water. But in what way can an absorption or ad-
sorption by the lipoids of the cell, of a host of different substances,
cause the development of the egg, and how can we explain those
methods in which no lipoid soluble substance is used?

Loeb had shown the similarity between methods of artificial mem-
brane formation and hemolysis, and many eggs segment after artificial
membrane formation. But in my opinion Loeb has not given a satis-
factory explanation of the mechanism of hemolysis. It seems to me
that the “‘membrane theory” of hemolysis, so admirably presented
by Stewart,” is a satisfactory explanation.

Lillie © advanced the view that the essential element in artificial
parthenogenesis is the increase in permeability of the plasma mem-
brane to COs., allowing the chief end product of oxidation to escape
and the rate of oxidation to increase, the more rapid oxidation causing
development. But Lillie has never published any determinations of
changes in permeability of the egg to anything except pigment, and
there is no certain proof that the escape of pigment is due to increased
permeability of the plasma membrane, as the pigment must first be
liberated from the chromatophores, in which it is held physically or
chemically.

I know of no method of determining changes in permeability of the
egg to COs, but have thought of five methods for detecting changes in

14 ‘TRAUBE: Biochemische Zeitschrift, 1909, xvi, p. 182.

© STEWART: Journal of pharmacology and experimental therapeutics, 1909,
i, p. 40.

© LILLIE: Biological bulletin, 1909, xvii, p. 188.

250 J. F. McClendon.

the permeability of the egg to electrolytes in general: 1. Electric con-
ductivity of masses of eggs; 2. Electric conductivity of individual
eggs as determined by destructive effects of the electric current-on the
cell; 3. Plasmolysis; 4. Chemical analysis of masses of eggs; 5. Mi-
crochemical analysis of single eggs. These will be considered in the
order given.

Brown ™’ concluded that the membrane of the Fundulus egg is prac-
tically impermeable to salts and water during the first eight hours,
and becomes most permeable after eighteen to twenty hours. Ap-
parently he refers to the thick membrane which is pushed out after
oviposition, but Sollmann '’ observed that the ‘‘yolk” swells in dis-
tilled water or one-fourth molecular cane sugar, obliterating the peri-
vitelline space, and thus indicating that the plasma membrane is less
permeable to salts than is the thick egg membrane or chorion (vitel-
line membrane of Sollmann).

Biataszewitz 1° found that the absorption of water by the unfer-
tilized frog’s egg increased five times for every rise of 10° C., and con-
cluded from this that heat increased the permeability of the plasma
membrane to water.

THe ELectric CONDUCTIVITY OF MASSES OF EGGs.

This work was done at the Tortugas Laboratory of the Carnegie
Institution. The experiments were made on board the yacht “ Phy-
salia”’ anchored off Boca Grande Key, twelve miles west of Key West,
Fla. I am indebted to Dr. W. R. Warren of Key West for the use of a
centrifuge, as the one provided was left behind. My thanks are also
due Dr. Alfred G. Mayer for the unusual facilities at my disposal.

The determinations were made by Kohlrausch’s method. A re-
sistance box of 15,000 ohms and a metre sliding resistance were used.
A number of difficulties arose in eliminating possible sources of error,
and these will be considered in order:

First. The procuring of sufficient quantities of suitable eggs to

17 Brown: This journal, 1905, xiv, p. 354.

18 SOLLMANN: This journal, 1904, xii, p. 112.

19 BIATASZEWITZ: Bulletin de l’Académie des Sciences de Cracovie, Math.
Nat., October, 1908.

On the Dynamics of Cell Division. 251

make accurate determinations. This was met by going to Boca Grande
Key, where the sea urchins, Toxopneustes variegatus and the Trip-
neustes esculentus, could be picked up by the ton in shallow water.
As the ripe eggs of the former species were more abundant, they were
used exclusively.

Second. The handling of the eggs with sufficient rapidity to insure
their being in normal condition. Washing the eggs repeatedly by
allowing them to settle in sea water requires much time, though it will
be shown later that for the first washing this is an advantage.” But the
time required for them to settle into a compact mass for the conduc-
tivity determination must be shortened as much as possible, as the
continued crowding of the eggs might produce abnormal effects. No
suitable conductivity vessel on the market could be placed directly
in the centrifuge.

I made a conductivity vessel at the Tortugas Laboratory. It con-
sisted of two glass tubes, the inner one fitting nicely into the outer
one. Owing to a very slight curvature of the tubes, a rotation of one
in the other would clamp them tightly together. The inner tube was
131 mm. long, 1o mm. inside diameter, and sealed at the lower end.
Two small glass tubes were placed longitudinally within the outer tube
and sealed into its upper end. One of these projected 25 mm. below
the other. Platinum electrodes 6 X 9 mm. were sealed in the lower
ends of the two smaller tubes. The electrodes were ‘‘platinized”’ with
platinic chloride solution containing a little lead acetate.

The advantages of this conductivity vessel were the comparatively
large surface area of the electrodes, 108 sq. mm. each; the distance
between them, 25 mm.; the small volume of eggs, less than 3 c.c.,
required to cover the electrodes, and the short time required for its
contents to reach the temperature of the thermostat. The electrodes
were plane and vertical, and hence could be pressed down into a mass
of eggs with the least possible disturbance of them. The inner tube,
containing the eggs, could be placed directly in the centrifuge, and
thus the eggs could be washed with sea water or other solutions without
removal from the conductivity vessel. By inserting the lower end of
the outer tube into a short, closely fitting test tube, the electrode could
be protected from drying while the inner tube was in the centrifuge.

* The eggs of Toxopneustes are of but very little greater specific gravity than
sea water and hence settle much slower than those of Tripneustes or Arbacia.

252 J. F. McClendon.

Third. The temperature of the thermostat, if constant, would on
some days be very far from that of the sea surface, which varied
greatly (from about 25°-30° C.), and the sudden change of the eggs
from one to the other might affect them in some undesirable way, as
they could be caused to segment by a change in temperature; also the
time required for the eggs to reach this temperature would be greater.
This was obviated by making the temperature of the thermostat about
one degree higher than that of the sea before each set of determinations
and then keeping it constant, within one tenth of a degree. The ther-
mostat held 20 litres of water, was closed at the top and well insulated
at the sides, stirred with a paddle attached to a rod going through a
small hole in the cover, and regulated by hand with a minute flame
beneath.

Fourth. The spaces between the eggs might vary. This was ob-
viated by centrifuging the eggs in the conductivity vessel and marking
their upper limit accurately in indelible ink with the finest drawing
pen, and before each reading centrifuging them again to the same
line.

The egg as it leaves the ovary is surrounded by an invisible gelati-
nous covering, which I have called the ‘jelly,’ since it shows similarity
to the jelly-like coat of the frog’s egg, a mucin which yields galacto-
samin (Schulz and Ditthorn). Loeb applies the name chorion to it.
This jelly seems to be a mucin which in sea water slowly dissolves.
The solution of the jelly is aided by weak or strong alkalis and very
weak acids, though it is coagulated by tannin and basic dyes, and
seems to contract (coagulate?) on addition of strong mineral acids.
Delage says it is lifted from the egg before the formation of the fertil-
ization membrane. It is possible that this appearance was due to
contraction caused by the dye used to make it visible.

If eggs bearing this jelly are put in the conductivity vessel and
centrifuged, and later mixed with sea water and centrifuged again,
much less force is required to precipitate them to the same level, and
they will slowly settle below this level by gravity while the eggs are
being brought to the temperature of the thermostat. This is due to
the washing away of some of the jelly. To prevent this occurrence,
the jelly had to be entirely washed off of them before they were first
placed in the conductivity vessel, a process accomplished by stirring
in large quantities of sea water and repeated centrifuging. The

On the Dynamics of Cell Division. 253

jelly could be removed completely from Toxopneustes eggs and with
more difficulty from Tripneustes eggs. Care was taken that no treat-
ment was used that would cytolyze even a few eggs, as cytolysis causes
swelling or disintegration which would affect the volume of the eggs
and therefore the spaces between them when they were precipitated
to a certain level.

The pushing out of fertilization membranes might affect the shape
of the spaces between the eggs and thus change the free paths of the
ions in the sea water filling those spaces. This was obviated by wash-
ing the eggs so long in sea water that no fertilization membranes
were pushed out when they were fertilized or treated with the solu-
tions used. This was tested by microscopic examination of control
eggs and of the eggs taken from the conductivity vessel after each
experiment. Such eggs develop.

It has been objected that a membrane was formed and not pushed
out. I found two methods of detecting membranes that lie so close to
the egg as not to be distinguishable with the microscope. If the egg
be plasmolyzed with a molecular solution of cane sugar, such mem-
branes are often if not always lifted from the egg. Harvey says the
fertilization membrane is relatively impermeable to sugar, and one
would suppose that sugar would push the membrane closer to the egg.
From many of my experiments it is evident that sugar will go through
the fertilization membrane, and Harvey has not stated the degree of
impermeability to sugar that he observed. The second and more
certain method is as follows: If an electric current of sufficient density
be passed through, the membrane will be lifted from the cathode end
of the egg.

It may be objected that the sugar solution and electric current
caused the membranes to be formed, and that they were lifted from the
egg in the usual manner. I can only answer to this that the same
sugar solution and electric current were tried on normal unfertilized
eggs and no membranes appeared.

Under the same conditions as those of the later conductivity ex-
periments, eggs did not form membranes that could be detected by
either of the above methods, and similar eggs developed. Harvey ™
says that after an egg stands in sea water twenty-eight hours and is
then fertilized, it becomes surrounded by a thick adhering membrane

21 HARVEY: Journal of experimental zoédlogy, 1910, viii, p. 365.

254 J. F. McClendon.

which, on cleavage of the egg, surrounds each blastomere. He calls
this a fertilization membrane, and maintains that fertilization mem-
branes are formed on all developing sea urchin eggs. Evidently in
the above case and in that of eggs placed in hypertonic or calcium-
free sea water he mistook the so-called “hyaline plasma layer” or
“Verbindungsschicht”’ for the fertilization membrane.” Harvey states
that he saw membranes on Hipponoe eggs on slightly “‘high focus.”
A membrane on the surface of a sphere could only be determined
positively with so high a power that the optical section was extremely
thin in comparison to the diameter of the sphere, and passed exactly
through the point of contact, with the sphere, of a tangent drawn
from the eye to the sphere. Hence there is only one focus, and “high”
or ‘“‘low” focus means out of focus. One need only try this “high
focus” on an air bubble or oil drop to see what appearances of ‘“mem-
branes” are thus obtained.

But it is impossible for me to see how a change in a surface film,
of immeasurable thickness, of the egg, thus forming a “‘membrane”
in close contact with the egg, can cause such a change in the conduc-
tivity of the inter-egg spaces as to account for the great differences in
the conductivity of unfertilized and fertilized eggs which I obtained.
I account for the change in conductivity by a change in the surface
film of the egg, allowing ions to pass through more easily. Probably
such a change would be accompanied by a visible change if micro-
scopic technique were sufficiently developed to detect it, but I would
not call this the formation of a new membrane; it is a change in the
‘plasma membrane,” a condensed surface tension film or haptogen
membrane.

The conductivity of the spermatic fluid and of the acidulated sea
water were slightly less than that of natural sea water, so that if they
replaced natural sea water between the eggs, they would cause a
slight decrease in the conductivity reading, but they were thoroughly
washed out with natural sea water before the readings were taken.

Fifth. The electric current passed through the eggs might alter
their conductivity. As no measurement of conductivity could be taken
before the current began to pass through, I cannot determine this
point. But the first reading and later readings taken at short intervals
were always the same provided the eggs had been centrifuged down

#2 See GoLpscumipT and Poporr, Biologische Centralblatt, 1908, xxviii, p. 210.

On the Dynamics of Cell Division. 255

so compactly that they did not settle further by gravity, and the
content of the conductivity vessel was at the same temperature as the
thermostat. By using a special induction coil with a rheostat, an
alternating current of such high frequency and such low amperage
was obtained that when passed through my finger from electrodes
wet with sea water, I could not feel it, yet this was the current used
in the experiments, and variations in it could easily be detected with
the telephone used in the experiments.

Sixth. The increased elimination of carbon dioxide by fertilized
eggs might cause an increase in conductivity of the sea water between
the eggs. To test this, the conductivity of a sample of sea water was
determined. It was then charged with CO, in a “‘sparklet syphon,”’
and no increase in conductivity could be detected with the electrodes
used in the experiments with eggs. Perhaps with electrodes specially
adapted to good conductors like sea water, a change could be detected,
but it would evidently be extremely small and incapable of account-
ing for the large differences observed in the experiments with eggs.

THE CONDUCTIVITY DETERMINATIONS.

The conductivity of one sample of sea water at 30° C. was found to
be .o61, while that of unfertilized eggs washed in the same water and
precipitated by gravity was .04655 at the same temperature. The
conductivity of the same water at 32° C. was .0624 and of the same
eggs at 32° C., .o469. At 26° the conductivity of another sample of
sea water was .05535, of spermatic fluid direct from testes, .0439, and
of unfertilized eggs precipitated with the centrifuge, .co2404. In this
case the conductivity of the eggs was about one twentieth that of the
sea water, although they still contained some of the latter between
them. The conductivity of the eggs would have fallen still lower if
all of the sea water had been pressed out from between them.

First lot of experiments. —In all of these preliminary experiments
the thermostat was kept at 32° C., which was at times very near,
but at other times very much above, that of the sea. The eggs
were precipitated by gravity. The supernatant sea water in the con-
ductivity vessel was pipetted off, and the vessel shaken, then read-
ings taken, until successive identical results showed that the eggs
were of the temperature of the thermostat, after which less than a

256 J. F. McClendon.

drop of sperma was added, the vessel shaken again, and a second
series of readings taken. The observed conductivities are given in
the following table:

1. Unfertilized eggs .05980

Fertilized eggs .16950
2. Unfertilized eggs 04480
Fertilized eggs 04835

3. Unfertilized eggs .04690
Momentarily heated .05600 %

The above figures show a great increase in conductivity on fertili-
zation, or momentary heating to a point that will cause segmentation.

Second lot of experiments. — In this and all subsequent lots of ex-
periments the thermostat was brought before each set of readings
to about sea temperature, the eggs were precipitated in the conduc-
tivity vessel to such a degree that they were not further precipitated
by gravity.

1. Unfertilized eggs at 27.5° C. 01524

Fertilized eggs ‘“ i 01627
2. Unfertilized eggs “ 28°C. .01076
Fertilizedeggs “ x .01266

Third lot of experiments. — As it was feared that the admixture of
but a fraction of a drop of spermatic fluid might raise the conduc-
tivity independent of fertilization, and as by this method only a small
per cent of the eggs were fertilized, in the third and fourth lots of ex-
periments the eggs were centrifuged in the conductivity vessel and
their upper level marked accurately, and the conductivity determined,
then they were mixed with sea water containing sperm or acetic acid
in the conductivity vessel, and washed by repeated precipitations and
precipitated down to the same level, before determining the conductiv-
ity of the developing eggs. By this method almost roo per cent of the
eggs could be caused to begin development.

1. Unfertilized at 26° C. .002404
Fertilized “ 7 .004320

23 The vessel was set a few moments in water at 45°, then returned to the ther-
mostat; this was shown by control to cause segmentation.

On the Dynamics of Cell Division. 257

2. Untertilized at 25.75° C. 004445

Fertilized af i .006340
3. Unfertilized at 25° C. .006523
Fertilized ‘‘ - .009544
4. Unfertilized at 26.25° C. .004900
Fertilized ‘ ¥ .006390
5. Unfertilized at 28° C. .008230
Fertilized . .009220
6. Unfertilized at 28.17° C. .006876
Fertilized 7 007298
7. Unfertilized at 26° C. .005620

After .006 normal acetic
acid in sea water 144
min. 26° C. .006000

Fourth lot of experiments, in which all precautions were taken. — In
these experiments the eggs were washed so long in sea water that
no fertilization membranes could be caused to push out.

1. Unfertilized at 29.5° C. .01182
Fertilized ‘“ a6 .01537
2. Unfertilized at 30° C. .01153

After .006 normal acetic
acid in sea water 114

min..30n'C; .0127
3. Unfertilized at 30° C. .00877
After acid sea water 1144
min 30° C. 00965

4. Unfertilized at 29.5° C. .005135
After acid sea water 114
min. 29.5° C. .00839

From the above experiments I have concluded that there is an in-
crease in electric conductivity of the sea urchin’s egg at the beginning
of development. The question now arises whether the resistance to
the movement of ions through the mass of eggs is the impermeability
of the plasma membrane or the presence of fat globules or proteid
granules within the egg, or the combination of the egg electrolytes
with colloids, forming poorly dissociated or poorly diffusible com-
pounds. I found by centrifuging them that there was as great a vol-
ume of fat globules and proteid granules in the sea urchin’s egg im-
mediately after, as there was immediately before, fertilization.

258 J. F. McClendon.

ON THE INTERNAL CONDUCTIVITY OF THE CELL.

The majority of my experiments on this subject were made at Cor-
nell Medical College.

Hober * has devised a method by which the electric conductivity of
the cell interior may be measured without breaking the cell wall. The
determinations cannot be made with great accuracy, but the results
on blood corpuscles clearly demonstrate that the conductivity of the
interior is many times greater than that of the corpuscle as a whole,
indicating that the greatest resistance to the current lies in the plasma
membrane.

Stewart made certain determinations which I take to indicate
that hen’s egg yolk is a very much poorer conductor than is a solution
of its salts made up to the same volume.

The yolk of the hen’s egg before it leaves the ovary forms the bulk
of a single cell. The yolk of an egg is often considered as “‘dead”’
material; but in conductivity experiments we cannot separate “liv-
ing” and “‘dead”’ portions of the cell. There is a small amount of
white yolk, but the major volume is yellow yolk.

The yellow yolk under the microscope presents a fluid matrix con-
taining large globules of another fluid of almost the same specific
gravity, viscosity, and refracting index as the matrix, the boundary
between the two fluids being the seat of little surface tension. Both
matrix and globules contain numerous fine granules. On the addition
of alcohol, under the microscope, a substance (or substances) in both
fluids disintegrates, setting free lipoids which appear as droplets, which
are blackened by osmic acid and colored by Sudan III. If one of the
large globules is watched closely as the alcohol is applied, fine, lipoid
droplets appear and grow and fuse to form larger drops, and some of
them may then migrate toward the periphery and fuse to form a lipoid
envelope surrounding the globule. The lipoid droplets appearing in
the matrix grow and fuse to form larger ones.

The yellow yolk darkens after the addition of osmic acid, the glob-
ules becoming darker than the matrix, but if alcohol and then osmic
be applied, the lipoid droplets thus formed, quickly become an in-

* Hoper: Archiv fiir das gesammte Physiologie, rg1o, Cxxxill, p. 237.
* STEWART: Journal of experimental medicine, 1902, vi, p. 257.

On the Dynamics of Cell Division. 259

tense black. Only the lipoid droplets are colored by Sudan IIT in 80
per cent alcohol.

The white yolk resembles the yellow yolk that has been treated
with alcohol in that it contains lipoid droplets. But as there is very
little white yolk in the hen’s egg before incubation, there are very few
lipoid droplets to impede the electric current. The lipoids in the yellow
yolk are probably bound up with proteids, forming combinations
which are disintegrated by alcohol, as indicated by the above
observations.

In order to determine to what extent the granules impede the electric
current, I precipitated them with the centrifuge. The large globules
cannot be separated from the matrix by this means. With small quan-
tities I was able to obtain the fluid entirely free from granules. It
forms 11/17 of the total volume, dissolves in dilute alkalis, and with
slight milkiness in dilute acids, and when shaken with water the
insoluble portion forms an emulsion or a coagulum resembling yeast
plants.

With large enough quantities to fill the smallest suitable conduc-
tivity vessel at hand, the precipitation was so slow that I feared de-
composition might commence before a granule-free fluid was obtained,
so I contented myself with the comparison of a portion containing a
very small per cent of granules with a portion containing a very large
per cent of granules.

At 25° C. the conductivity of the granule-poor layer was .00302
and that of the granule-rich layer .00o278, showing that the granules
impede the current to a great extent.

Since dilution with water breaks up many ion-colloid compounds, I
used this method to determine whether the electrolytes in the yolk
were bound up with colloids. The conductivity determinations at
25° C. are given in the table below:

Paced (Undiluted) (+ 1 vol. H,O) (+ 3 vols. H,O) (+ 7 vols. H,O)
: Vol. = 1. Vola: Vol. = 4. Nolo:
Granule-poor .00302 .00268 .00162 .00096
Granule-rich .00278 .00278 .00200 .00125

The above table shows that whereas the granule-poor layer de-
creases in conductivity on dilution with distilled water, at first slowly
and later slightly more rapidly (which may be partially accounted
for by the more rapid increase in ionization of inorganic salts at the

260 J. F. McClendon.

beginning than at the end of the series) the conductivity of the granule-
rich layer is not reduced at all by a dilution with one volume of H.O.
It may be said that this is due to the separation of the granules, thus
widening the conducting paths, and I have demonstrated that such
might occur in an emulsion of oil in soap solution, as shown by the
following table of conductivities at 25° C.:

Maternal (Undiluted) (+ 1 vol.H,O) (+ 3 vols. H,O)
j Vok= Vol. = 2. Voli A:
Soap solution 002490 .001460 000903
Emulsion of oil, containing
17 per cent soap solution .000434 .000434 .000335

But how are we to explain the fact that on dilution with one or more
volumes of water the conductivity of the granule-rich portion of yolk
is greater than that of the granule-poor layer, although the former
contains less of the fluid portion of the yolk? Evidently (since there
are no inorganic crystals in the yolk) some of the electrolytes must
have been bound up in the granules (either by adsorption or chemi-
cal combination) and liberated on dilution. Since the fluid portion of
the yolk does not entirely dissolve in water, the undissolved portion
may impede the current, but this would occur in the granule-rich as
well as in the granule-poor portion.

I doubt that Héber’s method of measuring the internal electric con-
ductivity of cells is sensitive enough to determine whether the increase
in conductivity of the egg is due to liberation of electrolytes in the
interior or to increased permeability of the plasma membrane of the
egg, but it shows, by exclusion, in case of the cells on which it was used,
that by far the greatest resistance to the current lies in the plasma
membrane.

Swelling (first stage of cytolysis) of sea urchin eggs causes a decrease
in the conductivity of the mass of eggs, as shown by the following de-
terminations of the conductivity:

1. Unfertilized eggs of Toxopneustes at 27.25° C. .01354
After addition of nicotine at 27.25° C. .01318
2. Unfertilized eggs at 32° C. 04850
After momentary elevation to about 50° C. at 32° .04780
3. Unfertilized eggs at 27.5° C. 01645
After momentary elevation to about 50° C. at 27.5° .01626
4. Unfertilized eggs of Tripneustes at 32° C. 04730

After shaking with fraction of a drop of chloroform at 32° C. .02286

On the Dynamics of Cell Division. 261

Microscopic examination showed that the addition of nicotine or
chloroform, or momentary elevation of temperature in the above ex-
periments, caused the eggs to swell. This could only take place by
the absorption of one or more constituents of the sea water between
the eggs.. If the salts of the sea water did not go into the eggs, the ab-
straction of H.O from the sea water would increase the concentration
of salts in the sea water remaining between the eggs, and might cause
a liberation of electrolytes (by dilution) within the eggs, in the latter
case causing increased conductivity of the egg interior without a cor-
responding decrease in conductivity of the inter-egg spaces. If the
membrane became freely permeable to salts, the swelling of the eggs
might increase, but should not diminish, the conductivity of the mass.
The fact that the conductivity decreased can only be explained by
assuming that the salts of the sea water entered the eggs and were ad-
sorbed to or combined with colloids, or that the membrane was very
poorly permeable to salts (though perhaps more permeable than the
normal egg) and the narrowing of the inter-egg spaces caused the de-
crease in conductivity. In fact, I think this can be taken as an indi-
cation that the egg is a poor conductor not so much because of the low
concentration of free electrolytes within it, but chiefly because the
electrolytes cannot easily pass the plasma membrane.

As no dilution of the contents (swelling) of the sea urchin’s egg
occurs at the beginning of development, it is improbable that a libera-
tion of the electrolytes within it, sufficient to account for the increased
conductivity, occurs. The only alternative is that the increase in con-
ductivity is due to an increase in permeability of the plasma mem-
brane to electrolytes.

THE ELEecTRIC CONDUCTIVITY OF INDIVIDUAL EGGS.

It is well known that cells may be killed or injured by the passage
of electric currents through the media containing them. The current
might affect them by raising the temperature, by the passage of ions
into or out of the cells, by the accumulation of ions of one sign that
are stopped by parts of the cell.

In which of these ways does the current affect the cell most de-
structively? The heating effect may be practically eliminated. If
electrolytes are transported into or out of the cell by the current, they

262 J. F. McClendon.

could also diffuse in the absence of a current, but the accumulation of
ions of one sign would not occur by free diffusion. Therefore I have
regarded the accumulation of ions impeded by the cell structures as
explanation of the destructive effects, and the destructive effects as
an indicator of the resistance to the passage of ions.

The experiments were made at the United States Bureau of Fish-
eries at Woods Hole, Mass. The r10-volt direct current from the
light circuit was used. Cylindrical non-polarizable electrodes of cop-
per in one-half molecular copper sulphate were plugged at their free
ends with absorbent cotton and connected to rubber tubes of 4 mm.
internal diameter and about one foot each in length, filled with sea
water. The free ends of the rubber tubes were plugged with absorbent
cotton, which was allowed to protrude sufficiently to conduct the cur-
rent to the sea water containing the eggs under a cover glass on a slide
on the microscope stage. The current was reduced by passage through
a 16-candle power light and further regulated by turning the screw of
a pinch-cock which was clamped on one of the rubber tubes. The
copper sulphate diffusing into the sea water in the rubber tubes re-
acted with the calcium carbonate, copper hydrate and calcium sulphate
being precipitated and carbonic acid being liberated. The copper
sulphate solution and sea water were renewed before each experiment.
Usually a piece of ash-free filter paper was cut the size of the cover
glass and a hole cut in its middle. This filter paper ring was placed on
the slide, and sea water containing eggs placed in the hole in the ring,
so that when the cover glass was placed on the preparation, the eggs
were contained in a cell which was freely permeable to ions at the

sides. At other times the eggs were mixed with sea water containing

enough cotton fibres to support the cover glass. Eggs of Arbacia punc-
tulata were used.

When the current is passed through the egg, the latter is affected
at that surface nearest the anode, as observed by Brown, who placed
the eggs in a molecular solution of urea. Changes in surface tension
are indicated by bulging or amceboid movements. The pigment sud-
denly leaves each of the chromatophores in turn and diffuses into the
cytoplasm in this region of the egg, which is turned a red or orange
hue (it is a deeper red if the chromatophores have been stained with
neutral red), showing that the reaction is not alkaline.* The anodal

* The pigment extracted from the eggs is red or orange in acid according to
dilution; it is violet or green and precipitates in alkali. If the eggs, or especially

On the Dynamics of Cell Division. 263

end of the cell absorbs water and swells, often a blister is formed and
masses of granular cytoplasm pass into the blister fluid and dissolve.
Gradually these changes extend from the anode end to the cathode
end of the egg, the egg swells enormously and may burst.

Very probably this disintegration commencing at the anode end of
the egg is due to the accumulation of anions which cannot pass the
plasma membrane. If the plasma membrane is poorly permeable to
anions in one direction, it is probably so in the other, and it may be
asked why they do not accumulate outside the cell at its cathode end.
The anions which are unable to enter the egg at its cathode end are
free to move around the egg and hence do not accumulate to form
as great a concentration as at the anode end.

Since no destruction of the egg of Arbacia, beginning at the cathode
end, was observed, we may conclude that the plasma membrane is more
permeable to cations than to anions. This is not true of all eggs, as I
observed the eggs of Hydractinea begin to disintegrate at the cathode
as soon as at the anode end. However, it is true of a number of living
cells?

If fertilized and unfertilized Arbacia eggs are placed in an isotonic
sugar solution containing little sea water, through which a current of
gradually increasing density is passed, the unfertilized eggs begin to
disintegrate, at their anode ends, sooner than the fertilized eggs do.
We may interpret this as indicating that the fertilized eggs are more
permeable to anions, which therefore accumulate in them to a less
extent, or the fertilized eggs are more permeable to electrolytes, which
therefore have passed out into the sugar solution to a greater extent,
and therefore the current passes through them less, than in case of the
unfertilized eggs.

I did not obtain the same results on eggs in sea water, but the un-
certainty of the material toward the end of the season prevented the
determination of the mode of action of the sugar solution. Possibly
the heating effect of the current in sea water increased the permeabil-
ity of the unfertilized eggs. Sea water is so much better a conductor
than the eggs that only a small per cent of the current passes through
the latter, and in order to produce visible effects on the eggs an enor-
the perivisceral fluid cells containing much pigment, are killed, the nuclei and

some other parts absorb the pigment and turn brownish purple.
*7 See VERWORN’S Physiology.

264 J. F. McClendon.

mous current must be passed through the sea water. It is known that
sugar solutions produce abnormal conditions in eggs, but these ex-
periments were made quickly after placing the eggs in the sugar solu-
tions. The nucleus does not begin to disintegrate as soon as the cyto-
plasm; this is in harmony with McCallum’s view that the nucleus
contains no free salts. The nucleus as a whole or the contained nucleo-
proteids migrate toward the anode.

PLASMOLYSIS WITH NON-ELECTROLYTES.

Osterhout has obtained shrinkage of marine cells in distilled water,
and thinks the action of sugar similar; 7. e., first the membrane is made
permeable and then the salts diffuse out and the cell contracts by
some non-osmotic force. But in the only animal cell in which he has
obtained this result there is first a swelling, with formation of blisters,
and later shrinkage, with the nucleus becoming homogeneous and
distinct, which, I think, denotes death and perhaps coagulation. Since
the Arbacia egg in an isotonic sugar solution does not swell first and
then shrink, I think this objection may not apply to my experiments.

The following tables show that fertilized (Arbacia) eggs shrink
more rapidly than unfertilized eggs in molecular sugar solutions,
which are calculated to have only slightly greater osmotic pressure
than the sea water at Woods Hole, where the experiments were made.
It appears that the plasma membranes of the fertilized eggs are more
permeable, allowing the salts to diffuse out of the eggs more rapidly,
thus lowering the internal osmotic pressure to a greater extent than
is the case with unfertilized eggs. Sollmann ** observed Arbacia eggs
contract in hypertonic, and swell in hypotonic, salt solutions.

In normal sea water fertilized are not smaller than unfertilized
eggs.?® Before the first cleavage the hyaline plasma layer forms, thus
taking material away from the more opaque portion of the egg, and
it might be supposed that the failure to include this layer in taking
the measurements caused the appearance of shrinkage, but such would
be the case also in the control in normal sea water, and furthermore
the measurements were taken before the hyaline layer was formed or
had reached visible thickness.

28 SOLLMANN: This journal, 1904, xii, p. III.
29 McCLENDON: Science, 1910, xxxii, p. 318.

On the Dynamics of Cell Division. 265

Fertilized and unfertilized eggs in a molecular solution of dextrose
were placed under the same cover glass, which was supported to pre-
vent compression of the eggs, and sealed to prevent evaporation. The
fertilized were distinguished from the unfertilized eggs by the presence
of the fertilization membrane. The eggs were observed in the order
in which they appeared in the field as the slide was moved so as to
observe the whole area under the cover glass once and once only. The
diameter of each egg in turn was drawn with the camera lucida, and
the drawings were measured later with a rule. In case an egg was
irregular, approximately its mean diameter was drawn.

The results of two series of measurements are recorded on page
266. In the first column of figures the diameter of the egg in the
unit used for all the measurements is represented. In the second and
third columns of figures the frequencies of the occurrence of fertilized
and unfertilized eggs of the diameters given in the same horizontal
line are represented. The fourth and fifth columns of figures repre-
sent a second series of measurements in the same manner.

The table shows that there is considerable variation in the
size of the eggs, but that the mean (and also the mode if the curve
were plotted) of the diameters of the fertilized eggs is less than the
mean of the unfertilized eggs. I did not succeed in making measure-
ments fast enough to determine the rate of plasmolysis.

CHEMICAL ANALYSIS OF MASSES OF CELLS.

Fertilized and unfertilized eggs may be placed in solutions differ-
ing from sea water, and the passage of substances into or out of them
detected by analysis of masses of the eggs. There are three sources
of error: 1. The presence of the jelly-like coverings on the eggs;
2. The fluid in spaces between the eggs; and, 3. The large surface for
adsorption.

I tried some preliminary experiments on yeast cells at a time when
suitable eggs could not be obtained. I found yeast and dextrose
placed in .3 molecular MgCl, eliminated CO: more rapidly than in .5
molecular NaCl or .325 molecular CaCl, all of which are calculated to
have approximately the same osmotic pressure. Also the CO, elim-
ination was more rapid in the magnesium solution than in a solution
of the same concentration of magnesium chloride with either of the

266 J. F. McClendon.

Frequency.

Diameter.
Fertilized. Unfertilized. Fertilized. Unfertilized.

111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144 '

ER) 1S ie* jie: CGN CNC) CN Ga) is Gone se a co ces ea tae

2
0
2
1
2
2
3
5
3
7
10
9
10
7
6
4
+
i
3
0
2
0
0
1

RPNNNHFPWWRNUNN FP NY WWNHNHWWNHNON OR.

|\F OOOO OCR FP EN WWHWHNTA YW OMWTAAUAWNH wee.

Mean diameter

On the Dynamics of Cell Division. 267

other salts in addition, or in a solution containing NaCl and CaCl, in
the same concentration as in the respective pure solutions, or in a solu-
tion containing all three salts, or in tap or distilled water.

The magnesium must have entered the cell or altered the permea-
bility of the plasma membrane to COs, sugar, alcohol, the enzyme, or
some other substance. In order to determine whether the magnesium
entered the cells, I took two blocks of compressed yeast of the same
volumes and weights and mixed one with H.O and the other with a
molecular solution of MgCl, for five hours, then washed each by rapid
precipitation in renewed H.O several times with the centrifuge. The
two lots were ashed and weighed with the results: control, ash = .0466
gm.; ash from Mg culture = .048 gm. Evidently the magnesium
did not enter the yeast to any great extent and probably acted on the
surface, increasing the permeability to some other substance.

Lyon and Shackell have analyzed fertilized and unfertilized eggs
placed in salt solutions, and obtained some results indicating that the
salts enter and leave the fertilized more easily than the unfertilized
eggs. They found an exception in the case of iodine. Iodine (in po-
tassium iodide solution) is absorbed by the unfertilized more quickly
than by the fertilized eggs.*?

I had intended to work along this line, but was forced to postpone
it until another season.

MICROCHEMICAL ANALYSIS OF INDIVIDUAL EGGS.

Lyon and Shackell *° and Harvey have concluded that certain dyes
enter fertilized more easily than unfertilized eggs. Loeb supposes that
the dye is chemically combined in the fertilized egg and merely in solu-
tion in the unfertilized egg. Unfortunately these dyes belong to the
class of substances which Overton found to most easily penetrate plant
cells, so that a demonstration that they more easily enter the fertil-
ized than the unfertilized egg does not necessarily indicate that the
same is true for electrolytes in general.

Harvey *' found that eggs became more permeable to NaOH after
being fertilized or treated with cytolytic agents.

3° Lyon and SHACKELL: Science, 1910, xxxii, p. 250.
31 HARVEY: Science, 1910, xxxli, p. 565.

268 J. F. McClendon.

THE MIGRATION OF THE CHROMATOPHORES.

The chromatophores of the egg of Arbacia contain a red substance
which I found to have an absorption spectrum similar to McMunn’s
echinochrome, at least in certain solvents. I have crystallized two
derivatives of the Arbacia pigment and perhaps the pigment itself,
and a chemical study of it is being attempted.

These chromatophores or pigment plastids show similarities to the
chloroplasts of some green plants. Similar plastids occur in the peri-
visceral fluid cells of Arbacia, where they are so closely packed to-
gether in the cytoplasm as to be separately distinguishable only on
careful observation. In some of the cells the plastids contain pigment
and in others they are colorless.

McMunn, finding that the spectrum of echinochrome in certain
solvents was changed by strong reducing agents, concluded that it was
respiratory in function. Griffiths * briefly states that on boiling with
mineral acids echinochrome is transformed into hemochromogen,
hematoporphyrin, and sulphuric acid, indicating a relation to
hemoglobin.

I separated the cells from about 50 c.c. of the perivisceral fluid of
Arbacia and mixed them with sea water to form 50 c.c. This suspen-
sion of cells, and 50 c.c. of sea water as a control, were exhausted under
an air pump for six hours, during the last half hour at practically
water vapor tension. While in the vacuum, the cells must have exerted
a reducing action on the pigment if it can be reduced. Each was then
shaken with air for thirty minutes in closed apparatus. The suspension
of cells had absorbed 1.25 c.c. of air and the control only 0.8 c.c., at
atmospheric pressure. The volume of oxygen used in oxidations in
the cells during the shaking was probably partly replaced by CO:
given off by them, but the difference of about half a cubic centimetre
does not demonstrate conclusively that the pigment combined with
oxygen. Under somewhat similar conditions dogfish blood absorbed
many times as much air as the perivesceral fluid of Arbacia.

The migration of the chromatophores in the egg is evidently not
always in the direction of greater oxygen concentration, but whether
it is ever a chemotropism toward oxygen I was unable to determine.

32 GRIFFITHS: Comptes rendus, 1892, Cxill, p. 410.

On the Dynamics of Cell Division. 269

In 1908 I observed movements of the chromatophores in the eggs
of Arbacia punctulata. As Roux had caused a whitening of the catho-
dal pole of the frog’s egg by passing an electric current through it, I
tried in 1909 and again in rg1o to move the chromatophores of the
Arbacia egg with the electric current. I observed that in the unfer-
tilized egg the chromatophores are distributed throughout the cyto-
plasm, but after the egg is fertilized or stimulated artificially the
chromatophores migrate to the surface.*

Harvey ™ says that the pigment comes to the surface within ten
minutes after fertilization, but I found that this process sometimes
required half an hour, by which time the cleavage spindle had formed.
At each cleavage chromatophores sink into the cleavage furrows of
the blastomeres. Just before the micromeres are formed the chroma-
tophores move along the surface of the blastomeres, away from the
micromere pole of the egg, so that after the resulting cleavage the
micromeres are practically free from pigment. Under abnormal con-
ditions there is a great massing of pigment in the cleavage furrow or
other regions of the surface or in the interior of the egg. The sinking
of pigment into the cleavage furrows and its retreat from the micro-
mere pole are probably due to surface tension changes as discussed
above, and perhaps the abnormal massing of pigment at one portion
of the surface is due to a local increase in surface tension.

‘““Membrane-forming” and parthenogenetic agents, even in concen-
trations too low to produce membranes or segmentation, cause the
pigment to come to the surface. If a few normal unfertilized eggs are
kept in a relatively large amount of sea water protected from evap-
oration, and oxygen is very abundant, it appears that there is more
pigment at the surface after twelve or more hours than at the begin-
ning of the experiment, but disintegration commences before all the
pigment has reached the surface. In an oxygen vacuum this did not
seem to occur. The pigment may all come to the surface in a stream
of washed hydrogen, but this may be caused by some impurity.

Fischel,® observing similar movements of pigment in the egg of
Arbacia pustulosa, concluded that the pigment was repelled by the
asters according to the forces described by Rhumbler as moving

°8 McCLENDon: Science, 1900, XXX, Pp. 454.
** HARVEY: Journal of experimental zodlogy, 1910, viii, p. 355.
%° FiscHeL: Archiv fiir Entwicklungsmechanik, 1906, xxii, pp. 526-541.

270 J. F. McClendon.

granules toward or away from asters in the cytoplasm.*® Biitschli and
Rhumbler have shown how the contraction of an area in a foam struc-
ture causes aster-like radiations around it, and Rhumbler has shown
that such radiations to a limited extent may occur around a rigid
sphere inserted into a foam or alveolar structure. Rhumbler assumes
that the concentration of the alveolar wall substance would increase
its surface tension, and that this increase toward the centre of the
aster would reduce the thickness of those alveolar walls perpendicular
to the astral rays, both of which assumptions have no facts of which I
am aware to support them,. On them rests Rhumbler’s explanation
of the movement of granules away from asters.

However, if those bodies which seem to be repelled by asters (chro-
matophores of Arbacia eggs, yolk platelets of frog’s eggs) lie within
or are larger than the largest alveoles, as I have observed to be the
case, aster formation might explain their repulsion. Rhumbler’s
theoretical asters were made of a central body and of alveoles of a
uniform size. If the alveoles were of different sizes, the largest ones
would seek the periphery of the aster.

Isectioned eggs that had been so treated artificially that all of the
pigment came to the surface but no segmentation occurred, and found
no asters, though perhaps asters had formed and disappeared.

After the passage of an electric current of a certain density and
duration through unfertilized eggs, some of them have their pigment
more abundant toward their cathodal surfaces. If the current exceed
a certain density, one by one the chromatophores toward the anodal
surface of the egg lose their pigment suddenly. When the current
was slowly and carefully increased just to the density required to
change the distribution of the pigment, no loss of pigment by the
chromatophores toward the anode could be observed, but it is mechan-
ically impossible to watch every chromatophore in the anodal region of
one egg. I found it possible to observe a single chromatophore for a
long time, and attempted, by noting its distance from the anodal sur-
face of the egg, to record its movements. Each time this observation
was attempted the chromatophore appeared to move, but its move-
ment was not constant in direction, and a considerable migration in
any one direction was not observed, except rarely in case the chro-

86 RHUMBLER: Archiv fiir Entwicklungsmechanik, 1806, iii, p. 527; 1890, ix,
pp. 32 and 63.

On the Dynamics of Cell Division. 275

matophore was very near the surface. In this exceptional case the
chromatophore moved along the surface, toward the cathode, which -
movement was probably due to surface tension changes. In the egg
just taken from the ovary the chromatophores are slightly more nu-
merous near the surface than in the interior, and when the current is
passed, this difference is increased. The passage of the current
causes the anodal surface of the egg to spread (the increased differ-
ence of potential between the two sides of the surface reducing the
surface tension), sometimes carrying the more superficial chromato-
phores along the surface toward the cathode. This is not a cataphore-
sis of the chromatophores, since they do not go in the direction of the
current, but is due to surface tension changes, and is therefore a
secondary effect of the current.

Fearing that the high viscosity of the cytoplasm might interfere
with the movement of the chromatophores by electric convection, I
centrifuged both fertilized and unfertilized eggs until the pigment
was massed at one pole of each, and passed the current through solu-
tions containing them. No orientation of the eggs to the potential
gradient occurred. I then tried to move the perivisceral fluid cells,
which are practically masses of chromatophores, by means of the
electric current, but my apparatus did not exclude all sources of error,
and this experiment was reserved for another season. The pigment
may be caused to leave the chromatophores in these cells by the elec-
tric current or by chemicals, to which agents these cells are much less
sensitive than are the eggs.

We have, then, no evidence that the chromatophores are electrically
charged.

Harvey *’ attempted to explain my observation that the chroma-
tophores come to the surface at the beginning of the development of the
egg, by assuming that there is a positive charge over the surface of
the egg until the commencement of development, when the surface
becoming permeable to anions causes a potential gradient between
the surface and centre of the egg. He further assumed that the chro-
matophores are charged negatively and migrate in the potential
gradient.

His evidence for the existence of the positive charge over the surface
of the unfertilized egg of Arbacia punctulata is the fact that it is not

37 HarvEY: Science, 1909, XXX, p. 604.

272 J. F, McClendon.

always spherical when it leaves the ovary. His evidence for the loss
of the charge is the fact that this egg rounds up more rapidly when it
is fertilized than when it is left in sea water without sperm. His evi-
dence for the negative charge on the chromatophores is the fact that
they come to the surface after development commences.

My observations indicate that the plasma membrane of the unfer-
tilized egg is less permeable to anions than to cations, which would
cause the appearance of the positive charge over the surface provided
some electrolyte whose undissociated molecules could not easily pass
the membrane was more concentrated in the egg than in the sea water,
or was produced with sufficient rapidity within the egg. Carbon diox-
ide might be this substance. However, my observations seem to indi-
cate that the permeability of the egg is increased suddenly (in less
than five minutes) on fertilization, in which case the positive charge
over the surface would be lost suddenly, and if the ions within the egg
were free to move, the potential gradient would be of momentary
duration, whereas the chromatophores require from ten to thirty
minutes to come to the surface. Before Harvey made this hypothesis
I had attempted, as described above, to move the chromatophores by
inducing a potential gradient, in order to determine whether they
were electrically charged. Harvey has yet to prove that they are
charged, and furthermore that they are negative, and that the poten-
tial gradient is of sufficient intensity and duration to move them to the
surface. I do not wish to be considered an opponent of his hypothesis,
but am merely searching for facts. Garbowski observed chroma-
tophores move toward the centrosomes.

ON THE CONTENTS OF THE ‘“‘ PERIVITELLINE”’ SPACE.

The assumption has been made by several observers that there
exists a colloid between the fertilization membrane and the egg. Here
the question arises, what is meant by the surface of the egg? The “‘hy-
aline plasma layer,” or ‘‘Verbindungsschicht,” which forms before
the first cleavage, is considered by some as part of the egg and by
others as a ‘‘membrane”’ outside of the egg. In this section I will not
include the hyaline layer in speaking of the egg, as under these experi-

mental conditions the surface of the hyaline layer (if such had formed)

On the Dynamics of Cell Division. 273

could not usually be distinguished, 7. e., the presence of this layer
could not be ascertained.

When an electric current is passed through the egg of Arbacia punc-
tulata having a “‘pushed-out”’ fertilization membrane, the latter is
bulged out toward the cathode, and the egg moved in the opposite
direction and pressed against the anodal portion of this membrane.
When the current ceases, the egg returns to the centre of the “peri-
vitelline space.” I first thought that this was caused by anodal elec-
tric convection of the egg, due to confined anions, but sometimes the
fertilization membrane bursts at its anodal pole and the egg passes
out, and should on this hypothesis continue its migration toward the
anode. But as soon as the egg is free from the fertilization membrane
it stops its migration, even though floating in a fluid of equal specific
gravity. Perhaps an invisible colloid having a positive charge fills the
perivitelline space, and its migration toward the cathode pushes the
egg in the opposite direction.

Loeb postulated a colloid in the perivitelline space as exerting an
osmotic pressure which pushed out the fertilization membrane. This
may be true, but. the membrane must harden in the expanded condi-
tion, for if it is burst by passage of the electric current or other means
it does not collapse, but remains spherical unless distorted by violence.

When the electric current causes bulging of the fertilization mem-
brane, the perivitelline space exhibits fine striations radially to the egg
or parallel to the current lines. Schiicking, Goldschmidt and Popoff,
Herbst, and others have described striations or fibres in the peri-
vitelline space, or around the fertilized egg, including the spaces
between the early blastomeres, usually under abnormal conditions.
These striations are probably due to tension of the colloid filling
the perivitelline space (including the hyaline plasma layer or
“Verbindungsschicht’’).

THE ACTION OF PARTHENOGENETIC AGENTS ON THE PLASMA
MEMBRANE.

Salts, acids, alkalis, shaking and thermal or electric changes might
alter the aggregation state of the colloids of the plasma membrane.
Fat solvents, alkaloids, glucosides, blood sera, soap and bile salts

Av.

274 J. F, McClendon.

might alter the aggregation state of the colloids, especially lipoids of
the plasma membrane.

Lillie *® found that pure solutions of sodium salts were effective as
parthenogenetic agents in the following series arranged according to
the anions: Cl<Br<NO,;<CNS<I. This order of anions is reversed
in the precipitation of lecithin, and a somewhat similar reversed
order of anions occurs in the salting-out of proteids.*® Hence it
appears probable that these salts act by virtue of their dissolving
power on the colloids of the plasma membrane.

Alkalis may act not only on the membrane, but by slow diffusion
into the egg favor oxidations, as an alkaline reaction favors oxidation
in general, and Loeb has shown that an alkaline reaction of the medium
is necessary for the normal oxidations in the sea urchin’s egg.

KCN or an oxygen vacuum may act by suppressing oxidation until
enough of the confined CO: can escape to raise the alkalinity within
the egg to such a point that when oxygen is readmitted oxidation may
proceed with sufficient rapidity to allow the development of the egg.

It seems probable that the undissociated molecules of carbonic acid
or COz can diffuse out of the egg at all times. How then could the
resistance of the plasma membrane to one or both or its ions so reduce
oxidation within the egg as to prevent its development? Perhaps the
per cent of CO, within the unfertilized egg is sufficient to lower the
alkalinity to such a degree that oxidation cannot proceed with suffi-
cient rapidity to allow development.

Loeb “*° finds that an oxygen vacuum or KCN reduces the toxicity
of certain poisons to unfertilized and to a greater extent to fertilized
eggs (poisons that affect fertilized in less concentration than unfer-
tilized eggs). He concludes that this cannot be explained on the per-
meability hypothesis (as the mere absence of oxygen would probably
not affect the permeability?). It may be that these poisons are toxic
because they increase the permeability of both fertilized and unfer-
tilized eggs, but since the fertilized eggs are more permeable before the
action of the poison, the additional increase in permeability is fatal
because oxidation is abnormally increased. Hence KCN or an oxygen
vacuum would be antitoxic.

38 Linu: This journal, ro1o, xxvi, p. 106.
39 HoEBER: Zeitschrift fiir Allgemeine Physiologie, 1910, x, B, p. 178.
40 LorB: Biochemische Zeitschrift, 1910, xxvi, p. 288.

On the Dynamics of Cell Division. 275

Loeb“ finds that after membrane formation a saccharose solution
of much lower osmotic pressure will cause the egg to develop than a
pure NaCl solution, which, he says, proves that the membrane is per-
meable to salts or ions, for the explanation requires that the egg salts
diffuse into the sugar in the former case or the NaCl diffuses into the
egg in the latter. This shows a certain degree of permeability of the
egg to electrolytes after “‘membrane formation,’ but proves noth-
ing as regards the normal unfertilized egg. It is not necessary to
postulate absolute impermeability, even of the unfertilized egg, to
electrolytes, in order to account for development by increased perme-
ability, and I have shown that an increase in permeability follows
the action of membrane-forming substances. Furthermore it has not
been demonstrated to my satisfaction that the action of the hyper-
tonic solution after membrane formation is purely osmotic.

Lyon has shown that the CO, and catalase elimination by the sea
urchin’s egg increases after fertilization, and Warburg, Mathews,
and Loeb and Wasteneys have shown that the oxygen absorption in-
creases. These changes might be due to an increase in permeability.

It is not supposed that an increase in permeability will cause any
cell to divide; growth is prerequisite to division. However, permea-
bility might influence growth. Growth is supposed to cause division
only when it affects the volume of the cytoplasm more than that of
the nucleus. The ratio of the cytoplasm to the nucleus in the egg
may be considered sufficient for a number of successive divisions, or
the ‘“‘true”’ cytoplasm may grow at the expense of the yolk after
each division.

** LoresB: University of California publications, Physiology, 1908, iii, No. 11,
Deore

ON ADAPTATIONS IN STRUCTURE
AND HABITS OF SOME MARINE ANIMALS OF
TORTUGAS, FLORIDA.

BY J. F. MCCLENDON,
Instructor in Histology, Cornell University Medical College, New York City.

2 plates, 1 text figure.

55

e

=e

a.

ON ADAPTATIONS IN STRUCTURE AND HABITS OF SOME .MARINE
ANIMALS OF TORTUGAS, FLORIDA.

By J. F. McCLenpon.

In June, 1908, at the laboratory of the Carnegie Institution of Wash-
ington, at Tortugas, Florida, I began the study of the habits of some reef
animals with a view to some comparative studies of behavior. The
results were written up in the Zoological Laboratory of the University
of Missouri.

It was found that many of these animals were thigmotatic and
remained in glass tubes rather than in the open. They also learned
to find the tubes when removed from them. Such was the case with
five species of the Alpheide, one of the Pontoniide, Typton tortuge
Rathbun, and Gonodactylus erstedii. All the anemones were thigmo-
tactic on their bases. These same animals were heliotropic. The Crus-
taceans were negatively heliotropic and the anemones kept their bases
from the light, while Cradactis variabilis Hargitt hid all but the tips
of the fronds and tentacles from the light. In removing its base from
the light, Stozchactis helianthus, which lives on coral heads, makes snail-
like movements similar to Metridium,! while Cradactis, which lives in
holes in decayed coral heads, crawls on its tentacles.

ON ADAPTATIONS OF SYNALPHEUS BROOKSI AND TYPTON
TORTUG-.

In lagoons between the reefs is found the loggerhead sponge, Hir-
cinia acuta, which grows to 3 feet or more in diameter, but is of no com-
mercial value. The passages in this sponge are thickly populated by
Synalpheus brooksi Coutiére. These Alpheids are thigmotactic and
negatively heliotropic and seldom come outside the sponge, which they
do only at night and then rarely leave its surface. The only other
animals seen in the interior of the sponge were a small species of Amphi-
pod and a Pontoniid. The Alpheids were several hundred times as
numerous as the Amphipods or Pontoniids. Near or at the surface crabs
and worms were sometimes found.

Both Alpheid and the Pontoniid, 7ypton tortuge, have the fourth
and fifth pairs of thoracic appendages pincer-like (plate 1, figs. 1 and 3).
In the Alpheid the fourth and in the Pontoniid the fifth pair are asym-
metrically hypertrophied. In the Alpheid the asymmetry is very great,
and the large chela can be snapped with such vigor as to produce a loud,
clicking sound. When this claw is removed its mate grows to replace

1 McClendon, 1906, On the Locomotion of a Sea Anemone, Biol. Bull. ro.
LY

58 Papers from the Marine Biological Laboratory at Tortugas.

it and the asymmetry is reversed, as first shown by Przibram. It iS
not known on which side the large claw develops first. I interpret Her-
rick’s records as demonstrating that the large claw develops first on the
left side in Synalpheus minus (Alpheus saulcy1).1 It was found in my
specimens about as frequently on the right as left side in both large
and small individuals. Of 50 taken at random, 22 had the large claw
on the left and 28 on the right. In another species Przibram found 40
individuals with the large claw on the left and 47 on the right.

The Pontoniid Typton tortuge, as was stated above, has the pincer-
like appendages of the fifth thoracic segment well developed. One of
these claws is much larger than the other, but the asymmetry is not as
great asin the Alpheids. Both of these claws are snapped with a sharp,
clicking sound. When the large claw is removed the small one grows to
take its place, as in the Alpheids.

The two animals do not perhaps resemble one another as much in
general coloration as in general form, though the color varies so much in
both animals that these differences are not at first noticeable. The color
darkens with age. The Alpheid varies from the color shown in plate 1,
fig. 1, toa light brown. Specimens with a claw like fig. 2 may bea dull
cream or light brown in general color. The nerve cord and some other
organs may be surrounded by red pigment cells. Yellowish, brownish,
or reddish glands in thorax or abdomen may show through.

The Pontontid Typton tortuge varies from the color shown in fig. 3
to an almost colorless condition, or to a light red or a pale bluish. The
large claw of the pale specimens is often paler than the small claw in
fig. 3. After the large claw has been removed the small one grows to
take its place, but for some time retains more or less its general form and
color. Often yellow, brown, or green glands show through in the thorax
and abdomen.

As these animals pass their entire adult existence in the dark or
dim light, it is improbable that their color is of much significance in their
struggle for existence; hence it would not be fixed by natural selection.
The fact that their eyes are not degenerate might indicate that they
sometimes come near the mouths of the passages in the sponge. Per-
haps they are forced out when the sponge becomes overcrowded, but
I doubt that many of the larger ones would find another sponge before
they were eaten by fish. Neither form was found in any other habitat,
though Herrick records the Alpheid from reef rocks as well as loggerhead
sponges in the Bahamas.

The Alpheid has large eggs, few in number, attached to the swim-
merets of the female. The metamorphosis is abbreviated, and in some
cases omitted. The young remain attached for a time to the mother,
but perhaps always leave the sponge and live a short pelagic life before
finding another sponge. The female Pontoniid deposits numerous small
eggs on the swimmerets. These hatch into small larve which lead a
comparatively long pelagic life before acquiring the form and habits
of the adult.

? Brooks and Herrick: The Embryology and Metamorphosis of the Macroura.
Mem. Nat’l Acad. Sci., 5, 1891.

’

Adaptations in Structure and Habits of Certain Marine Animals. 59

I studied the habits of these animals as well as I could in dim light
in the cavities of pieces cut from the sponge. They appeared to behave
normally, whereas in glass tubes in brighter light they remained motion-
less. Both animals explore the cavities of the sponge with cautious
movements unless disturbed, in which case they snap their claws. The
Alpheid advances, using its large claw as an antenna and protector.
Its antennz can be extended about as far forward as the large claw.
When meeting an Alpheid or a Pontoniid it may try to squeeze past or
it may snap its claw. When placed in glass dishes Alpheids cut one
another to pieces, but this is seldom if ever done in the narrow passages
of the sponge.

The Pontoniud Typton tortuge advances, using both claws as an-
tenne, the antennz being very much shorter than the smaller claw. It
spreads the claws apart and waves them about, thus exploring the cavity
in front of it. On meeting another it behaves as the Alpheid, except that
it may snap either or both claws. Both animals try to squeeze through
small openings. The chele of Typton sometimes show what appear
to be claw marks. As their claws are more slender, hence more easily
grasped and less powerful than those of the Alpheids, it is to be expected
that they would show claw marks first in case both species snapped with
equal frequency.

Both animals appeared to eat from the walls of the cavities in the
sponge, but I did not determine whether they ate the sponge itself or a

sediment deposited on it. I did not determine whether they ate one

another in the sponge, but they were so numerous that it seems strange
that they received sufficient oxygen. The Alpheids are sometimes in-
fested with a parasitic isopod, Bopyrus, in the gill cavity.

I do not intend to discuss here the origin of the form or habits of
these animals, but it seems to me that we have here a convergence both
in form and habit. It is probable that similarity in form and habits
made both animals better suited to living in the same habitat, 7.e., the -
sponge, and that accidentally finding the sponge they remained there.
However, this does not explain why the young at the end of pelagic life
aiways (or at least usually) select the loggerhead sponge. There are
numerous Alpheids living in holes in the reef rocks, and certainly they
are more closely related in form and general habits to Synalpheus brooksi
than is Typton.

This Synalpheus and the Typton select the sponge not because it
has holes in it in which they can hide, but on account of some more
specific quality, such as taste (smell), color, or outward form. Or when
some individuals of these species have established themselves in a sponge
the others may be attracted to it by a social instinct (which may not be
disproved by the fact that they destroy one another when placed under
unnatural conditions). The isolation of these animals in the loggerhead
sponge is an example of what Gulick calls habitudinal segregation and
may have been a factor in the evolution of the species.

Since the Alpheids occur in far greater numbers than Typton we
might suppose the former to be much better adapted to living in the
Sponge than the latter. However, although JT ypton produces more

60 Papers from the Marine Biological Laboratory at Tortugas.

eggs, it has a much longer pelagic life than the Alpheid and is much more
likely to be eaten or swept out to sea by the tides, where it can not find
a sponge when the proper time comes.

The smallest loggerhead sponges I found would not live in a large
aquarium with running sea-water more than 2 days before the water
began to get foul within the passages in the sponge and the Alpheids
and Typton began to die. Field observations were very limited. These
and other difficulties restricted the investigation to its present limits.

ON ADAPTATIONS OF THE REEF ANEMONE, CRADACTIS VARIABILIS.

Cradactis variabilis Hargitt is an anemone about an inch or two in
length when expanded, living in holes in old coral heads or reef rocks.
Besides the tentacles, which are few in number and arranged as in Sagar-
tia, long outgrowths called fronds extend from the region bearing the
tentacles (plate 1, figs. 4, 5; plate 2, figs. 8,9, 10). The animals may be
a moss-green or brown in general color, but the tentacles are always
paler and often colorless and transparent at their tips. The fronds may
or may not be branched, and may end simply or in pale knobs, as in
plate 1, fig. 4, or in curious “‘eyes,”’ as in fig. 5.

These anemones are usually found in cavities in old coral heads that
communicate with the exterior by a number of passages about half an
inch or more in diameter. The anemones are attached near enough to
these passages to extend the tips of the fronds to the exterior (plate 2,
fig. 7). This extension is caused by heliotropism of the fronds. One
mistakes them at first for sea-weed, although they do not resemble any
particular kind of sea-weed that I have found growing on the reefs.
The tentacles are extended about as far as and sometimes a little farther
than the fronds, but the fronds tend to conceal the tentacles. At night
the fronds are contracted and the tentacles remain extended; therefore
it is probable that the fronds are not necessary as breathing organs.

If a bit of crab meat is held near the passage through which the
Cradactis is extended no response is obtained. But if one of the fronds
is touched with the meat the tentacles are extended toward it, while the
frond touched may contract slightly. In order to observe the food-
taking more minutely, some of the anemones were taken from the rock
and allowed to attach themselves to the bottom of an opaque dish filled
with sea-water. When a bit of crab meat is placed on the end of a ten-
tacle it adheres and the tentacle and one or more adjacent ones are bent
down and the food placed on the mouth and pressed there. Immediately
many or all of the tentacles are pressed on the food, hiding it from view
until it is swallowed. The fronds may contract more or less during
the process. Cradactis sometimes swallows filter paper placed firmly on
the mid-region of a tentacle or on the disk, but not when placed on the
end of a tentacle. This may be a question of degree or extent of stimu-
lation. It disgorges the paper within 10 minutes. It rejects bits of shell,
etc., placed on the disk or tentacles.

India ink placed in the water near the anemone showed ciliary
currents running towards the tips of the tentacles and fronds, and on

Adaptations in Structure and Habits of Certain Marine Animals. 61

the disk running towards the mouth. A secretion sticks the particles
together. These currents are useful on both fronds and tentacles in the
rejection of particles, and on the tentacles in the placing of food in the
mouth, the food being carried to the tip of the tentacle before it is placed
in the mouth. ;

When disturbed by light falling on the base, it sometimes moves
with snail-like motion (like Metridium) a short distance, but the tentacles
catch hold of the substratum on all sides. The tentacles and column
sometimes perform writhing movements. More often the animal bends
over to one side and catches hold of the substratum with the tentacles,
with or without previously elongating the column, the fronds contract-
ing slowly all the while. It then loosens the base, walks on its tentacles
to a new place (plate 2, figs. 11, 12), bends over and attaches the base,
and lets go its hold with the tentacles. This method of locomotion is
much more rapid than that of Metridium, but could not be used if the
Cradactis did not live in holes, as it might otherwise be washed away
by the currents that constantly sweep over the reefs.

The resemblance of the fronds to sea-weed leads one to suppose that
they act as lures or in hiding the Cradactis from its prey (anemones
being unpalatable are usually not in need of protection). The fact that
the fronds are heliotropic and contracted completely at night is in har-
mony with this view. I did not

cut them off to see whether the AW
anemone would live and repro- AEE w fe VEY

duce as well without them. The
cavities containing the Cradactis
a b

: 47 \
are inhabited by other animals, * eX
especially a small black crab, ox d
b Fic. 1.—Cradactis variabilis. a, Planula just escaped
and one might suppose that the aie ccelentric See Re mother, oral esentd)
side uppermost. , The same, second day, seen
fronds protected the tentacles of from oral side; pigment arranged radially. ¢, The
same, thir ay. : e same, fourth day; ten-
the anemone from the legs of the tacles elongating and septa becoming distinct. The
crabs that crawled over it. The mouth should be elongated in the plane of sym- ~

metry.
crabs are active at night in the

least light in which they can be seen (their black color making them
hard to see in the holes in the rock). In case they are normally active
at night the fronds would serve as a protection from the crabs only half
of the time. The anemones sometimes grasp the crabs and hold them
until they wrench themselves loose, which they invariably do in a short
time. Perhaps the anemone gets part of its food as particles dropped
from the crabs’ mouths.

Cradactis develops to the planula stage in the ccelenteron of the
mother. On being released, the planula swims around for a few hours
(text fig. 1, a) and attaches itself (b) by the smaller end. It gradually
develops a mouth and tentacles (b-d). When first liberated, the planula
has 8 mesenteries, and 8 tentacles develop soon after. Individuals were
seen with 8, ro, 12, 14, 16, 18, 20, 22, 24, 26, 28 and more tentacles.
From this one might conclude that the tentacles (and mesenteries)
appear in pairs, but they were often observed to appear in sets of four,
Symmetrical in relation to the oral plane. The first pair of fronds appear

62 Papers jrom the Marine Biological Laboratory at Tortugas.

in the 20-tentacle stage as outgrowths of the body-wall just beneath
the tentacles and with their axis perpendicular to the oral plane. The
second pair of fronds appear in the 28-tentacle stage or later.

SUMMARY.

(1) Convergence in structure and habitat is the cause of commen-
salism between an Alpheid and a Pontoniid living in the loggerhead
sponge.

(2) Abbreviation of its pelagic life accounts for the numerical super-
sedence of the Alpheid. -

(3) The weed-like outgrowths or fronds of a reef anemone, Cradactis,
probably hide it from its prey.

(4) Cradactis is kept just within the mouths of cavities in reef rocks
by the combined action of negative heliotropism of its base and positive
heliotropism of the fronds. The fronds are entirely contracted in the
absence of light.

(5) The fronds possess the sense of taste but do not carry food to
the mouth.

(6) Cradactis moves from place to place by walking on its tentacles,
a phenomenon sometimes seen in Hydra.

DESCRIPTION. OF PLATES.

(Figures 4 and 5 were redrawn by Mr. Kline, fig. 6, from life, by K. Morita,
otherwise the drawings and photographs are the author’s.)

PARE ae

. Synalpheus brookst Coutiére.

Chela of same species to show different coloration.

. Typton tortuge Rathbun commensal with the above.

. Cradactis variabilis Hargitt.

. The same, showing another variety in color and shape of fronds.
. Cradactts variabtlis Hargitt, X 2.

QDunfPWD

PrAnE so.

7. A portion of an old coral head showing the fronds (f) of Cradactis protruding
from the cavities.

8-10. Cradactis variabilis, showing varieties in shape of fronds.

11,12. Cradactis variabilis, walking on its tentacles, with detached base toward
the observer.

MS CLENDON

Coutiére.

PLATE I.

to show different colorations.
sal with the above.

: BMeisel it
4. Cradactis variabilis Hareitt.

5. Cradactis variabilis Hargitt.

6. The same, showing a third variation.

McCLENDON

PLATE 2

Fig. 7. A portion of an old coral head showing fronds (f) of
Cradactis protruding from the cavities.

Figs. 8-10. Cradactis variabilis showing varieties in shape of fronds.

Figs. 11-12. Cradactis variabilis walking on its tentacles with detached
base toward the observer.

PUBLICATIONS

OF

Cornell University

me O/CAL COLLEGE

-

met DIES

FROM THE

Department of Anatomy

@>

POLUME BF
IQII-12

mow 6K ORK CIY

CONTENTS

Being reprints of studies issued in 1911 and 1912.

1. THE FATE OF OVARIAN TISSUES WHEN PLANTED ON DIF-
FERENT ORGANS. By Charles R. Stockard.
Arch, f. Entw.-Mech., Vol. XXXII, 297-307 and 3 plates.

2. THE RETICULUM OF LYMPHATIC GLANDS.
By Jeremiah §S. Ferguson.
Anat. Record, Vol. V, 249-260 and 10 figures.

3. THE APPLICATION OF THE SILVER IMPREGNATION METHOD
OF BIELSCHOWSKY TO RETICULAR AND OTHER

CONNECTIVE TISSUES. By Jeremiah S. Ferguson.
Am. Jour. Anat., Vol. XII, 277-296 and 13 figures.

4. A PRELIMINARY NOTE ON THE RELATION OF NORMAL
LIVING CELLS TO THE EXISTING THEORIES OF THE
HISTOGENESIS OF CONNECTIVE TISSUE.

By Jeremiah §. Ferguson.
Biol. Bull., Vol. XXI, 272-279 and 2 figures.

5. ON THE STROMA OF THE PROSTATE GLAND, WITH SPECIAL
REFERENCE TO ITS CONNECTIVE TISSUE FIBERS.
By Jeremiah S. Ferguson.
Anat. Record, Vol. V, 541-546 and 1 plate.

6. THE TEACHING OF VISCERAL ANATOMY, OR ORGANOLOGY.
By Jeremiah S. Ferguson.
Jour. A.M.A., Vol. LVI, 1544-1546.

7, DUODENAL DIVERTICULA IN MAN. By Wesley M. Baldwin.
Anat. Record, Vol. V, 121-140 and 4 plates.

8. THE PANCREATIC DUCTS IN MAN, TOGETHER WITH A
STUDY OF THE MICROSCOPICAL STRUCTURE OF THE
MINOR DUODENAL PAPILLA. By Wesley M. Baldwin.

Anat, Record, Vol. V, 197-228 and 12 figures.

9. A METHOD OF FURNISHING A CONTINUOUS SUPPLY OF
NEW MEDIUM TO A TISSUE CULTURE IN VITRO.
By Montrose T. Burrows.
Anat. Record, Vol. VI, 141-144.

to. THE RELATIONSHIP BETWEEN THE NORMAL AND PATHO-
LOGICAL THYROID GLAND OF FISH.
By J. F. Gudernatsch.
Johns Hopkins Hospital Bulletin, Vol., XXII, May, 1911.

Ir. HERMAPHRODITISMUS VERUS IN MAN.
By J. F. Gudernatsch.

Am. Jour. of Anat., Vol. XI, 267-278 and 7 figures, $ plates,

ine

14.

15.

rz

19.

. EIN FALL VON HERMAPHRODITISMUS VERUS HOMINIS.

By J. F. Gudernatsch.
Verh. des VIII, Internat. Zool. Kongress zu Gratz, 570-574.

THE OSMOTIC AND SURFACE TENSION PHENOMENA OF

LIVING ELEMENTS AND ‘THEIR PHYSIOLOGICAL

SIGNIFICANCE By J. F. McClendon.
Biol, Bull., Vol. XXII, 113-162.

AN ATTEMPT TOWARDS THE PHYSICAL CHEMISTRY OF.

THE PRODUCTION OF ONE-EYED MONSTROSITIES.

By J. F. McClendon.
Am. Jour. Physiol. Vol., XXIX., 289-297.

THE INCREASED PERMEABILITY OF STRIATED MUSCLE TO

IONS DURING CONTRACTION. By J. F. McClendon.
Am. Jour. Physiol., Vol. XXIX, 302-305.

. DYNAMICS OF CELL DIVISION—III. ARTIFICIAL PARTHENO-

GENESIS IN VERTEBRATES. By J. F. McClendon.
e Am. Jour., of Physiol., Vol. XXIX, 298-301.

HOW DO ISOTONIC SODIUM CHLORIDE SOLUTIONS AND
OTHER PARTHENOGENIC AGENTS INCREASE OXIDA-
TION IN THE SEA URCHIN StEGG:

By J. F. McClendon.
Jour. of Biol. Chem., Vol. X, 459-473.

. A NOTE ON THE DYNAMICS OF CELL DIVISION. A REPLY

TO ROBERTSON. By J. F. McClendon.
Arch, f. Entw.-Mech., Vol. XXXIII, 263-266.

EIN VERSUCH, AMOBOIDE BEWEGUNG ALS FOLGEERSCHEI-
NUNG DES WECHSELNDEN ELEKTRISCHEN POLAR-
ISATIONSZUSTANDES DER PLASMAHAUT ZU _ ER-

KLAREN. By J. F. McClendon.
Arch. f. die ges, Physiologie, Bd. 140, 271-280.

The Fate of Ovarian Tissues when planted
on different Organs.
By

Charles R. Stockard,

Cornell Medical School, New York City, U.S.A.

With 2 figures in text and Plates XI—XIII.

Eingegangen am 20. Januar 1911.

It is a well known fact as has been shown in the case of the
ovary and thyreoid that an entire organ may be transplanted from
one individual to another without discontinuing to grow or function.
GuTHRIE and CasTLe have found that fowls and guinea pigs with
transplanted ovaries may ovulate and reproduce in an entirely normal
manner. MEISENHEIMER has conducted a most elaborate and beauti-
ful series of experiments to show that although the sex glands may
easily be transplanted from one to the other sex in caterpillars yet
the moths into which these caterpillars metamorphose show the
typical secondary sexual characters of their original sex, not being
affected by secretions from the transplanted bodies. All of these
experiments indicate that there is no antagonistic action towards
the organs of a different individual, even of the opposite sex, when
planted into the body of another individual of the same species of
animal.

Yet we seem to face a different proposition when considering —
the transplantation of portions of organs or tissues from one animal
to another. In most cases such pieces of organs or tissues live and
may actually grow for a time but invariably they cease to grow
and finally disappear entirely. In this way normal tissues differ
from malignant growths which continue to grow sometimes even
more actively after transplantation.

The Fate of Ovarian Tissues when planted on different Organs. 299

It is also true, and I think a point of importance, that the
ability of a tissue transplant to live and grow depends largely upon
the kind of tissue on which it is planted. The indiscriminate in-
jections of tissue emulsions and tissue pulps of both adult and
embryonic tissue as sometimes used in the experimental study of
cancer, are most unreliable and rarely give results owing to the hit
and miss method employed. All tissue transplants must be care-
fully made and a circulation established by grafting in minute
blood vessels, before deductions are to be drawn from the reactions
which follow. It is evident that if the transplant is not properly
nourished during the first hours or days it will begin to undergo
degenerative changes which will in all cases effect its future behavior.

With these points in view the questions arise: First, do certain
transplanted tissues survive equally well when planted on any organ,
or do they survive longer and better on certain organs than on
others (provided of course that the attachment and circulation is
equally good in all cases)? Secondly, if they do survive better on
certain organs what relationship exists between the tissues and these
organs and what is the cause of their better survival ?

Leo Logs and Appison showed a few years ago that when
guinea pig tissue was planted into other species of animals and
into other guinea pigs, that the tissue always grew better in guinea
pigs than in any other animal; and it was further indicated that
the tissue survived for a shorter time the more distantly related
was the species into which it was transplanted. Thus, as might
have been expected there is a specific reaction on the part of the
body of an animal to the transplanted tissues of other species of
animals. .

The present experiments, although of a preliminary nature,
bear upon the question of a resistance or antagonism between the
tissues of one organ and those of another in either the same indi-
vidual or other individuals of the same species. And also the further
question, of antagonism between different tissues, or different cells,
in similar organs.

The animals used in these experiments were guinea pigs and
the common salamander Diemyctylus viridescens. The tissue chosen
for transplantation was that of the ovary since it is composed of
Wo so entirely different classes of cells, the stroma tissue and the

erm cells or ova, and further as an organ is so interestingly related
the testis of the male. Pieces of the ovary of guinea pigs were
20*

300 Charles R. Stockard

transplanted into the testis and into the body wall and liver tusses .

of the male. They lived better when planted into the testicle and
here the artificially established circulation seemed more efficient than
in other organs. Nevertheless, all of the experiments with ten
guinea pigs were unsatisfactory since in no cases did the trans-
planted tissue live sufficiently long or well to allow valid comparisons.
Ripsert, Luparscu, Levin, LOEB and others have all had similar
experiences in transplanting the tissues of mammals. The tissues
may grow for a short while but soon stop and ultimately all dis-
appear. Inflamatory conditions are also commonly produced in the
guinea pigs if not operated upon with great care.

With the salamanders the experiments were much more satis-
factory, the ovarian tissue is easily transplanted and grows and lives
for several months, in many of the cases, and undergoes changes
so slowly and uniformly as to permit careful study and comparisons.

Thirty individuals were employed in the experiments and por-
tions of ovary in various degrees of maturity were planted on the
liver, lungs, kidney, stomach and body wall of the same individual
and on the testis, stomach, kidney, body wall, lungs and liver of
male salamanders. The ovarian tissue grew equally well on similar
organs of the male as upon those of the female, showing that there
-s no marked individual reaction against the tissues of other spe-
cimens, even though of the opposite sex. The most favorable of
all transplants, as will be considered below, was that of ovary
tissue on the testis.

The transplanted tissue in all cases was carefully attached to
the new organ with the finest silk or gut fibre suture and neigh-
boring blood vessels were dissected out and embedded within and
around the tissue, the entire operation being performed under the
binocular microscope. The blood vessels in almost every case
readily sent out branches and supplied the new tissue.

Ovarian tissue on the testis.

The tissue from the ovary, as stated above, was most persistant —

and successful in its growth when planted upon the testis. A portion
of the ovary containing ova or germinal epithelium and stroma tissue
was planted in a pocket or slit cut in the testis, a branch of the
spermatic artery was carefully dissected loose and placed around
the ovarian mass which was fastened to the testicle by a silk or

The Fate of Ovarian Tissues when planted on different Organs. 301

gut-fibre suture. Ten such operations were entirely successful. The
ovarian piece was well nourished and the ova continued to grow,

in most cases each ovum
having a plexus of capil-
laries about it as is shown
in the camera drawings
-(Figs. 1 and 2).

After forty-one days
the entire plant is in an
apparently normal condi-
tion (Figs. 1 and 2). There
are no indications of de-
generation in either the
egg cells or stroma (PI. XI,
Fig. 1). At no time does
there seem to be a ten-
dency for the cells of the
testicular stroma to mi-
grate in, or replace the
ovarian stroma cells. The
separation between the

A camera drawing showing part of an ovary planted on the
testis of a salamander 41 days after the operation. 0 Ova,
all well supplied by capillaries indicated in black lines;
C scars resulting from the cuts made during the operation.

testicular and ovarian tissue remains distinct. As will be mentioned

The same specimen shown from the opposite side. The heavy lines indicate the rich supply

of blood vessels, be,

below, this is in contrast to the reaction of other tissues to the
Ovarian transplant (see Figs. 2 and 4, Plates XI and XII).
The egg cells seem to be unable to continue their growth as

302 . Charles R. Stockard

they are highly laden with yolk and need certain conditions for
their maturation and further multiplication or development, The
yolk granules then begin to loosen apart, and later globules are
formed by the fusion of groups of granules, these globules become
scattered throughout the ovarian tissue as is seen by comparing
transplants of different ages (Plate XII, Figs. 3 and 4, and Plate XIII,
Figs.6 and 7). These globules persist for as long as seven months
and might for much longer. It thus seems that the stroma cells
do not tend to appropriate yolk bodies as food. This inability of
these cells to use yolk as food, or to absorb it, is necessary that
the egg cells may have an opportunity to accumulate or form yolk
in the presence of the stroma tissue. Yolk granules disappear much
earlier in ovarian transplants on other organs since the cells of
these organs migrate into the ovarian piece and dissolve or absorb
the yolk grains.

After the dissipation of the yolk, as for example, in a seven
months’ transplant (Plate XUI, Figs. 6 and 7) the ova cells can no
longer be distinguished, the entire piece seems of the stroma cell
type with yolk globules scattered through the tissue. In rare cases,
however, masses of yolk globules are localized and probably re-
present the persistant remains of an egg cell (Plate XII, Fig. 3). The
pigment granules of the egg are lost and cannot be identified in the
old plants.

The ovarian stroma persists and is well preserved in the trans-
plants of seven months’ duration. They would doubtless have
lasted much longer if specimens could have been kept after this time.

It appears then, that there is little antagonistic response between
two such organs as the ovary and testis and that the tissues of
these organs do not tend to replace or destroy one another, thus
they may live and grow side by side. The ova persist perfectly
for one or two months and then undergo retrogressive changes
probably on account of having passed the period at which they
should have matured or undergone some important modification.
The stroma of the ovary, however, is not attacked by the testicular
stroma and may survive in an apparently perfect condition for longer
than seven months, which is as far as the observations extended.
Ovarian transplants on all other organs had entirely disappeared —
long before, neither the ova or stroma being able to exist in contact
with the tissue cells of such organs.

The Fate of Ovarian Tissues when planted on different Organs. 303

Ovarian tissue on the liver.

Ovarian tissue when planted on the liver with a blood supply
established persists longer than on any other organ except as dis-
cussed above on the testis. The ovarian tissue persists equally well
on the liver of the same individual or on that of a male of the same
species of salamander. In no case out of fifteen successful trans-
plants on the liver did the blood vessels branch and supply the
ovarian piece so efficiently as did the branches of the spermatic
artery. This was due to the fact that no artery of the liver could
be so nicely placed about the piece as could the spermatic of the
testis. The circulation of the liver-plant, however, was very good
and easily sufficient to have supplied the piece as was shown by
the length of time it persisted. The difference in circulation does
not account for the earlier fate of the ovarian piece on the liver,
but this difference was due to the different way in which the liver
tissue or cells themselves reacted to the strange tissue. While there
was no tendency on the part of the testicular stroma to encroach
upon the ovarian tissue and replace its cells by wandering testicular
cells, the liver very soon reacted in such a manner.

The ova and stroma cells lived well on the liver for several

weeks though they did not grow very much. After this time, how-
ever, the cells of the liver had encroached and migrated into the
ovarian tissue apparently replacing and destroying the tissue before
it. The ovarian piece decreases in size and the ova gradually loose
their yolk and finally themselves disappear. Fig. 2, Plate XI and
Fig. 4, Plate XII show an ovarian piece planted upon the liver; it
will be noticed that the liver tissue extends into the ovarian part
and many liver cells have migrated far into the transplant, the
typical pigment spots of the liver are seen in the ovarian graft in
Fig. 4, Plate XII. These figures are photographs of a 42 day trans-
_ plant and may be compared with Fig. 1 Plate XI which is a trans-
_ plant of about equal duration on the testis.
Transplanted on the liver the ova show all indications of de-
generation and breaking down while on the testis they are in a
normal healthy condition. The large blood vessel, bv, at the base
of the transplant on the liver as well as smaller vessels shown in
the sections of the transplanted tissue would indicate that the piece
was sufficiently nourished and did not degenerate on account of a
poor blood supply.

304 Charles R. Stockard

The liver cells, then, show a kind of antagonistic action against —
the ovarian tissue which is not shown by the cells of the testis.
This we may speak of as the antagonism between two different
organs or the antagonism between tissues of different organs. A more
pronounced antagonism, mentioned before as shown by Leo LOEB
for transplanted tissues and well known from many haemolysis ex-
periments, is that which exists between the tissues or parts of animals
of different species, specific antagonism.

Ovarian tissue on lung, kidney, stomach and body wall.

Pieces of ovary planted upon the lungs, kidney, stomach or
body wall of the same or of another individual often live for a short
time before being absorbed but usually disappear within a week or
ten days after the experiment, only three out of more than fifty
such transplants lived as long as 45 days. These three were almost
completely replaced and would have soon disappeared.

A fair circulation may be established for the transplant on any
of these organs, yet the ovarian tissues seem unable to maintain
themselves in such an environment and both ova and stroma
begin to degenerate and are readily replaced or absorbed by the
cells and tissues of the supporting organ.

It is difficult, from the observations at hand, to state whether
or not certain of these organs are more antagonistic to ovarian
tissue than others. The wall of the stomach is an unfavorable
place to make a transplant, but the body wall would seem favor-
able since a good circulation is easily obtained, yet the tissue
readily breaks down and disappears in either place. Transplants
on or within the delicate lung tissue often break away but do not
thrive even when successfully made, and pieces of ovary on the
kidneys readily disappear.

Summary and Conclusions.

From these experiments on thirty salamanders it would seem
that the behavior or fate of transplanted tissues depends largely
upon the nature of the organ upon which the tissue is transplanted.
Ovarian tissue grows and lives incomparably better when trans-
planted upon the testis than upon any other of the body organs
experimented upon.

The next most favorable ground for this tissue was upon the
liver, although here the liver cells soon begin to encroach upon the

The Fate of Ovarian Tissues when planted on different Organs. 305

ovarian mass replacing and absorbing its cells. On other organs
the ovarian tissue undergoes degeneration and absorption within a
very limited time. .

Ovarian tissue planted upon the testis persisted with the stroma
in good condition for more than seven months. On the liver ovarian
tissue was found still to contain ova and stroma after more than
45 days. While on the body wall, lungs, kidney and wall of the
stomach the tissue disappears within about two weeks, only three
indications, in more than fifty such transplants, were found after
45 days and these had almost disappeared.

It is very important, therefore, to realize that there is a marked
difference between the reactions of certain tissues to others, and all
transplants should be made in the most careful manner between
organs of as near as possible a similar type. The introduction of
mixtures or emulsions of adult or embryonic tissue into different
parts of the body is a very unreliable method and one likely to
give most contradictory results, depending upon the proportion of
certain tissues present which successfully plant, and upon the organ
of the host into which the tissues are placed or happen to reach.

Just as there is a specific reaction between the tissues of an-
imals of different species which tends to prevent the growth of
foreign tissue when planted in their bodies, there seems to be, from
this preliminary series of experiments, a reaction between the tis-
sues of different organs of the same or different individuals which
causes transplanted tissue to exist to better advantage on one, usu-
ally a more closely similar organ, than on another.

Zusammenfassung und SchluBfolgerungen,

Nach den vorstehenden Versuchen an 30 Salamandern hiingt das Verhalten
oder das Schicksal transplantierter Gewebe anscheinend weitgehend von der
Natur des Organs ab, auf welches sie transplantiert wurden. Ovarialgewebe
wiichst und lebt bei Transplantation auf den Hoden unvergleichlich viel besser,
als auf irgend einem andern der in den Versuchen gewiihiten Kirperorgane.

Den nichstgiinstigsten Grund fiir dieses Gewebe stellte die Leber dar, ob-
gleich hier schon die Leberzellen auf die Ovarialmasse iiberzugreifen und deren
Zellen zu ersetzen und zu absorbieren anfangen. Auf andern Organen unterliegt
_ das Ovarialgewebe bereits innerhalb einer sehr beschrinkten Zeit der Entartung
und Absorption.

Auf den Hoden iiberpflanztes Ovarialgewebe erhielt sich mit dem Stroma
mehr als 7 Monate lang in guter Verfassung. Auf der Leber fand sich Kier-
Stockgewebe noch nach mehr als 45 Tagen im Besitz von Eiern und Stroma.

206 Charles R. Stockard

Wiihrend auf der Kérperwand, den Lungen, den Nieren und der Bauchwand
das Gewebe ungefiihr im Laufe zweier Wochen zu verschwinden pflegt, fanden
sich im ganzen zweimal Anzeichen desselben, unter mehr als 50 derartigen Trans-
plantationen, nach 45 Tagen, und auch diese bereits im Verschwinden begriffen.

Es ist daher sehr wichtig, festzustellen, da hier ausgeprigte Unterschiede
in den Reaktionen gewisser Gewebe auf andre bestehen, und alle Transplan-
tationen sollten daher so sorgfiiltig als méglich zwischen Organen von miglichst
nahestehendem Typus vorgenommen werden. Die Einfiihrung erwachsener oder
embryonaler Gewebe in Gestalt von Mixturen und Emulsionen in verschiedene
Teile des Kirpers ist eine sehr unzuverliissige Methode, geeignet, zu ganz
widersprechenden Ergebnissen zu fiihren und abhiingig von dem Vorhandensein
eines entsprechenden Betrages erfolgreich iiberpflanzbarer Gewebe, endlich von
dem Organe des Wirts, in welches die Einpflanzung stattfindet, oder welches
sie zufillig erreichen. P

Gerade so, wie es eine spezifische Reaktion zwischen den Geweben ver-
schiedenartiger Tiere gibt, welche das Wachstum eingepflanzten fremdartigen
Gewebes zu verhindern strebt, so scheint nach dieser vorliufigen Versuchsreihe
auch zwischen den Geweben verschiedenartiger Organe desselben oder verschie-
dener Individuen eine Reaktion zu bestehen, welche einem transplantierten
Gewebe vorteilhaftere Existenzbedingungen auf einem, gewdhnlich einem niiher
verwandten, Organ verschafft, als auf einem andern.

(Ubersetzt von W. Gebhardt, d. 6. II. 1911.)

Literature cited,

CastiE, W. E., On the Nature of Mendelian Factors. Read before Am. Soc.
of Naturalists. 1909.

Gururin, ©. C., Further Results of Transplantation of Ovaries in Chickens.
Journ. Exp. Zool. V. 1908.
Levin, L., Cell Proliferation under Pathological Conditions with especial Refer-
ence to the Etiology of Tumors. Journ. Med. Research. VI. 1901.
Logs, L., und Appison, W. H. F., Beitriige zur Analyse des Gewebewachstums.
Il. Transplantation der Haut des Meerschweinchens in Tiere verschiedener
Species. Archiv f. Entw.-Mech. XXVII. 1909.

MEISENHEIMER, J., Experimentelle Studien zur Soma- und Geschlechtsdifferen-
zierung. Erster Beitrag. Jena, Fischer, 1909.

Rous, P., Comparison of Conditions that Regulate the Growth of Transplanted
Tumor and Transplanted Embryo. Journ. Am. Med. Assoc. LIV. 1910.

Explanation of Plates,

All figures are microphotographs of sections through transplants of ovarian

tissue of various ages upon the testis and liver of the male salamander, Die-
myctylus viridescens.
Plate XI.
Fig. 1. A 41 days old ovarian graft upon the epididymal end of the’ testis.
The ova and ovarian stroma are still in normal condition. Ov ovary,
Ep Epididymis.

Pi Grohe ee,

es

oe

The Fate of Ovarian Tissues when planted on different Organs. 307

Fig. 2. An ovarian mass of about the same age planted on the liver. The two
sections are equally magnified and it is noted that the ovarian parts planted
on the liver are much reduced the ova being very small and almost all of
the yolk of the egg cells has been lost. Ov ovary, Li liver.

Plate XII. :

Fig. 3. A highly magnified section of ovarian tissue after seven months exis-
tance on the testis. An ovum, 0, is shown in which the yolk granules
have massed into globules.

Fig. 4. A higher magnification of the portion of ovary shown in Fig. 2 plate XI
which has been planted upon the liver of a male for 42 days. The liver
cells and liver pigment are shown to be encroaching upon the ovarian
mass. © ova, bv large blood vessel at the base of the transplanted tissue.
Compare the ova, 0, with that of the seven month transplant in Fig. 3.

Plate XIIT.

Fig. 5. A portion of ovary, ov, that has been living for seven months upon the
testis, Hp. All of the ova have disappeared but the stroma is still in good
condition.

Fig. 6. A higher magnification of the section of ovary in Fig.5. Seattered
throughout the stroma are seen yolk globules, y, which represent the
remains of the once large egg cells.

Fig. 7. A highly magnified section from another specimen of ovary after seven
months upon a testis. Here also the yolk globules, y, are scattered through
the stroma.

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Reprinted from THE AnatomicaL Recorp, VoL. 5, No.5
May, 1911

THE RETICULUM OF LYMPHATIC GLANDS

JEREMIAH 8. FERGUSON

Cornell University Medical College, New York City

TEN FIGURES

The silver impregnation method of Bielschowsky' in its newest
form has with some modifications been applied to the study of
tissues other than those of the nervous system for the staining
of whose neurofibrils it was originally devised. Maresh? and
Studnicka? seem to have been the first to appreciate its wider
applicability. Levi‘ slightly modified the method for use
with connective tissues, and his publication stimulated various
Italian observers to a study of the collaginous and reticular
tissues in a considerable variety of organs. Ciaccio, Cesa-
Bianchi, Alagna, Balabio, Favaro, and others have put forward
such publications, and the method has been shown to serve as a
fairly distinctive stain for the peculiar fibres of reticular tissue.
This method applied to nature tissues blackens only the reticular
tissue and shows beautifully its distribution.

The inequality in the distribution of the lymphocytes in lym-
phatic glands with formation of denser areas is well known.
The areas of great density are: 1, the cortex as compared with
the medulla; 2, the follicles; 3, the cords. The increased density
of the cortex is not solely due to the presence of follicles within
it, for the intervening areas are of greater density than much of
the medulla. In the follicles the periphery is of greater density
than the central portion. This is due to the presence of the

1Bielschowsky and Pollack, Neurol. Centralbl., 1904, Bd. 23, 387.
2Centralbl, f. allg. Path., 1905, Bd. 16, 641.
*Studnicka, F. K, Zeitschr. f. wis. Mik., 1906, Bd. 23, 414.
‘Levi, G., Monitore Zool. Ital., 1907, Anno. 18, 290.
249

250 JEREMIAH S. FERGUSON

germinal centers of Flemming. In the medulla the leucocytes
in the cords are densely packed as compared with those in the
intervening sinuses. While the facts regarding the crowding of
leucocytes have been generally recognized, the somewhat simi-
lar distribution of the reticulum has not been so frequently ob-
served. Silver blackened sections of lymphatic glands show
that the reticulum, like the lymphocytes, is crowded at certain
points. The denser areas are found in the cortex, the medullary
cords, at the periphery of the follicles, and about the blood-vessels
and trabecula. The condensation of reticulum beneath the cap-
sule on the borders of the peripheral sinus, and at the boundary
line between the cords and trabecula on the one hand and the
cortical and medullary sinuses on the other, is so pronounced
as to form a distinct membrane. Of two comparable areas it
often happens that the denser the lymphoid cells the denser the
reticulum. Thus, the reticulum of the cortex is denser than that
of the medulla, that at the periphery of a follicle is denser than
in its center, that of the medullary cords is coarser and often
denser than that of the sinuses: of course so general a rule is not
without exceptions, notably in that the cortical tissue between
the follicles is frequently provided with a very dense and firm
reticulum.

Ciaccio® has already called attention to the increased density
of the reticulum in the periphery of the follicle as compared with
the broad meshes and secant fibres of the germinal centers. This
perifollicular plexus of Ciaccio is very characteristic. It consists
of course fibres arranged to form peculiar lozenged-shape meshes.
Ciaccio states that in the active follicles of the spleen and lym-
phatic glands the intrafollicular network is rare, while in the
inactive it is nearly as dense as the perifollicular plexus. One
must however call attention to the possible error resulting from
the examination of sections whose vlanes pass through only the
peripheral portions of certain follicles (fig. 2). In such cases the
apparently active follicles show a dense network throughout a
considerable portion of their diameter but this net is obviously

5Anat. Anz., 1907, Bd. 31, 594.

RETICULUM OF LYMPHATIC GLANDS DA

All figures, unless otherwise stated, were drawn with the aid of the Edinger
projection apparatus and the magnification accurately measured. All sections
from which figures were made had been stained by the method of Bielschowsky.

Fig. 1 Follicles at the periphery of a lymphatic gland of a dog. Aod. P..,
adventitial plexus; Pf.P., perifollicular plexus; S. M., subcapsular membranes.
x 200, reduced } in reproduction.

© ty WS =
8

PesdeaSer cts
Pos f pee
“se Cases Z

: cy... eet
2 fe, y= en Se

Fig. 2 Two adjacent follicles in the cortex of a lymphatic gland of man. The
section passes only through the surface of the follicles and apparently shows a
dense intrafollicular plexus which is, however, in reality a surface view of the
perifollicular plexus. The coarseness and density of the plexus is apparent on
comparison with the intrafollicular plexus shown in fig. 1. XX 256, reduced } in
reproduction.

252 JEREMIAH S. FERGUSON

that of the peripheral plexus, the germinal center lying outside
the plane of the section. This is clearly shown in many of my
sections both in the lymphatic glands and in the intestine, spleen
and other lymphoid organs. The intrafollicular plexus of a
lymphatic nodule rarely approaches the density of the perifol-
licular, and some of those whose intrafollicular plexus showed
the closest net have been follicles which in axial section showed a
very distinct germinal center of Flemming.

Ciaecio (loc. cit.) has also called attention to the perivaseni
adventitial plexus of reticulum occurring in lymphoid organs. I
can corroborate his observations in so far as I find that such an
adventitial plexus surrounds all of the vessels within the lympha-
tic gland (figs. 1, 6 and 7). I desire to call attention, however,
to two other plexuses which have not hitherto been described as
formed by the reticular fibres. These plexuses are often so dense
as to form reticular membranes; they are found, the one, peri-
cordal, limiting the surface of the medullary cords and so forming
a wall for the lymphatic sinuses of the medulla, ‘and the other a
double membrane, the subcapsular, at the surface of the gland
beneath the capsule where it forms limiting walls for the peri-
pheral lymphatic sinuses.

A closer examination of the peripheral sinuses shows that
throughout a considerable portion of their extent they lie between
two distinct membranes of pure reticulum against which the
endothelium is applied. The outer subcapsular membrane
(O.S.M., fig. 3) is in contact with the capsule and its fibres to
some extent intertwine with the innermost collaginous fibres of
the capsule. Here and there, especially where it passes from the
surface of the capsule to the trabecula, the outer subcapsular
membrane may be partially detached from the collaginous tissue
upon which it elsewhere lies. At each trabeculum its fibres are
continued into the interior as well as upon the surface of the
fibrous septum, so that the trabecula contain throughout a con-
siderable proportion of reticulum, whereas in the capsule this
tissue is confined to the innermost layer of the membrane.

The inner of the two subcapsular reticular membranes is formed
by a condensation of the reticular network of the cortex of the

RETICULUM OF LYMPHATIC GLANDS . 253

trab oS LSM Ee p.
| —
er ee ae ™~ Sarceg it stl os
= SSS
NS arte
abe, oe 4 = te Vee Cte y
Ton \ Trae, Bens

Fig. 3 The peripheral sinus and its reticular subcapsular membranes from a
lymphatic gland of adog. Cap., capsule of the gland; 7.S.M., inner subcapsular
membrane; O.S.M., outer subcapsular membrane; trab., trabeculum. X 229,
reduced 2 in reproduction.

organ, and since it represents a condensation only, it is as one
must expect, much more evident at some points than at others.
Here and there it forms an almost continuous line along the cor-
tical surface of the organ (I.S.M.., fig. 3) while elsewhere one may
find areas where the condensation of reticulum is so slight that
in transections a membrane can scarcely be discerned.

In the peripheral sinuses, between the two reticular membranes,
the reticulum forms a loose network of delicate fibres and very |
broad meshes. The fibres of this net become directly continu-
ous with those of the reticular membranes.

In the medulla a reticular membrane occurs on the surface of
each of the cords, where it forms a limiting wall for the medullary
lymph sinuses. Certain lympho-glandulae in which the cords
are numerous and characteristic show continuous lines of reticu-
lar membranes on the surface of each cord (fig. 4). Here and
there the inner subcapsular membrane may be reflected inward
along the trabecula to become continuous with the pericordal
membranes (fig. 5B) so that the peripheral sinus passes directly
into the medullary. But more frequently the inner subcapsular
membrane is lost in the cortical reticulum in the vicinity of the
trabecula, the fibres of the outer wall of the sus pass into the

254 JEREMIAH S. FERGUSON

Fig. 4 Photomicrograph of two medullary cords of a dog’s lymphatic gland;
each is bounded by a distinct pericordal membrane. The small blood-vessels
within the cord show well the adventitial plexus. 250,reduced % in reproduc-
tion.

trabecula and within the gland become frayed out, as it were,
the fibres being continuous with those of the cortical reticulum:
in this case the peripheral sinus directs its flow of lymph into
the dense cortex, through which lymph must percolate in order
to reach the medullary lymph sinuses.

A third series of reticular membranes is formed about the walls
of the blood vessels by the adventitial plexuses of Ciaccio, and
similar adventitial or peritrabecular plexuses (pericordonal plexus
of Balabio) about the nonvascular as well as the vascular portions

RETICULUM OF LYMPHATIC GLANDS 255

of the trabeculae. In the vascular trabecula of the lymphatic
glands this plexus forms no part of the wall of artery or vein
but lies in the substance and upon the surface of the fibrous
trabeculum, the fibres of the plexus being continuous with those

Fig. 5 Two views of the pericordal plexus. A, surface view. At the point a,
the pericordal plexus is seen in tangential section and shows its membraneous
character; at the points b, the same membrane or plexus has been cut in transection.
Cd., medullary cord; S, medullary sinus. From a lymphatic gland of a dog.
x 400, reduced } in reproduction.

B, a portion of the cortex of a lymphatic gland of man, near the hilus. The
medullary sinus, S, leads directly from the subcapsular sinus about the trabeculum,
T, to the medulla of the gland. Af. Lym., afferent lymphatic; Ef. Lym., efferent
lymphatics; Cap. capsule of the gland; V, vein, making its exit through the con-
nective tissue of the hilus. The sinuses are bounded by the pericordal plexus.
x 60, reduced } in reproduction.

of the trabeculum. This is well shown in fig. 6, in which the col-
laginous adventitia of the artery and of the lymphatic vessels is
seen well within the blackened trabecular reticulum.

256 JEREMIAH S. FERGUSON

ey ee

Fig. 6 The adventitial plexus about the arteries in a vascular trabeculum in
the medulla of a lymphatic gland of man. The plexus lies just outside of the
tunica adventitia. > 220, reduced 4 in reproduction.

Balabio® states (p. 137) that the adventitial plexus of the blood
vessels has not the importance in the lymphatic glands that it
hasin the spleen. I can verify this statement so far as it concerns
the arteries, the periarterial plexus in the spleen being much
heavier than in the lymphoglandulae; but the same is scarcely
true of the veins (fig. 7). The small venules which Calvert’ has
described at the periphery of the follicles (figs. 1 and 8)stand out
prominently in the lymphatic glands because of their coarse,
close-meshed, adventitial plexus. With the exception of those
vessels lying within the vascular trabecula I find the thin-
walled veins of the spleen very deficient in those adventitial
fibres which blacken with silver.

Ciaccio (loc. cit.) dismisses the lymphatic capillaries with the
statement that they have a structure similar to that of the blood
capillaries. On p. 600 he says: ‘‘I capillari linfatici presentano

SAnat. Anz., 1908, Bd. 33, 135.
7Calvert, W. J., Anat. Anz., 1897, Bd. 13, 174; and Johns Hop. Hosp. Bull.,
1901, vol. 12, p. 177.

RETICULUM OF LYMPHATIC GLANDS . 257

Fig.7 Periadventitial plexus about the wall of a medullary vein in a lymphatic
gland of man. X 255.

. . 3 ts
A *#\ -verule

adv.pler

Fig. 8 Lymphatics and venules from the capsule and cortex, respectively,
of a lymphatic gland of a dog. A, afferent lymphatic vessels in the capsule; B,
venules in the adjacent cortex of the same section; adv. plex., adventitial plexus;
af. lym. ves., afferent lymphatic vessel. X 225.

quasi la stessa struttura dei capillari sanguigni, differendo da
questi soltanto per la forma, larghezza e disposizione.”’ From
my observations of the lymphatic vessels I am led to believe,
however, that the volume of reticulum in the adventitial plexus
is considerably less in the lymphatics than in veins of correspond-

JEREMIAH 8S. FERGUSON

QO

25

ing size. This is specially well shown if one compares vessels
somewhat larger than capillaries, e.g. the venules at the periphery
of the follicles and the afferent lymphatics in the capsule, (fig.
8), or such intratrabecular lymphatics as are shown in fig. 5B.
Within the mass of lymphocytes which forms the parenchyma
of the organ the reticulum is everywhere found. At the periphery
in the cortex and within the cords of the medulla the network is
formed by course fibres with polygonal meshes of relatively
small diameter Within the sinuses of both cortex and medulla
the fibres are much finer and the polygonal meshes broader so

Fig. 9 A medullary sinus in transection, showing its very delicate network
of reticular tissue. From a lymphatic gland of man. X 255.

that in the sinuses the total volume of reticulum must be only
a small fraction of that in the denser portions of the gland.
The broadest mesh is that in the germinal centers of follicles,
the finest fibres those of the sinuses. The shape of the mesh is
typically lozenge-shaped in the perifollicular plexus of Ciaccio, and
polygonal elsewhere, though here and there the polygonal spaces
are considerably elongated in the directions of stress or of lymph

RETICULUM OF LYMPHATIC GLANDS ~ 259

—— —— —_ ie

y r) f / vA

Se |

@ 2 °% oy,
oe? %e

@ ete" Ss )

Fig. 10 The reticulum at the periphery of a lymphatic gland of man; the
meshes are elongated in a direction perpendicular to the capsule. Cap., capsule
of the gland; 7ZSM, inner subcapsular membrane; OSM, outer subcapsular

membrane; Perip. Sn., peripheral sinus. X 625, reduced 4 in reproduction.
Camera lucida.

flow; this is most noticeable at the periphery of the cortex where
beneath the subcapsular membranes the meshes are elongated
in a direction perpendicular to the surface (fig. 10). This ar-
rangement is confined to the intervals between the cortical
follicles for wherever the surface of a follicle comes into contact
with the inner subcapsular membrane of the peripheral sinus the
meshes of the perifollicular plexus appear to be flattened against
the reticular membrane and the network of reticulum thus ren-
dered more dense than elsewhere.

bo
(op)
(=>)

JEREMIAH S. FERGUSON
SUMMARY

The lymphatic glands are everywhere pervaded by a reticulum
which blackens with silver impregnation by the method of Biel-
schowsky. ‘This reticulum is condensed to form more or less
complete reticular membranes bounding both cortical and med-
ullary sinuses. Hence one finds these dense plexuses or reticu-
lar membranes in several locations, viz., subcapsular (inner and
outer wall of the peripheral lymphatic sinus), pericordal about
the medullary cords, peritrabecular (equivalent to the ‘peri-
cordonal’ of Balabio) and adventitial (where they surround the
small naked blood-vessels).

The reticulum forms a net of close meshes in che periphery of
the follicles (perifollicular plexus of Ciaccio), in the cords a cordal
plexus, and in the peripheral portion of the cortex a cortical
plexus. The meshes are broad in the subcortical zone, still
broader in the cortical and medullary sinuses, and broadest in
the germinal centers of active follicles. The form of the mesh
is lozenge-shaped in the perifollicular plexus, sometimes elongated
in the cortical plexus, and elsewhere polygonal.

The reticulum is everywhere a continuous mass of stroma, the
fibres of both reticular membranes and plexuses being continu-
ous with each other throughout the gland.

Reprinted from THE AMERICAN JOURNAL OF ANATOMY, VOL. 12, No. 3
November, 1911

THE APPLICATION OF THE SILVER IMPREGNA-
TION METHOD OF BIELSCHOWSKY TO _ RE-
TICULAR AND OTHER CONNECTIVE TISSUES

1. THE MATURE TISSUES

J. S. FERGUSON
From Cornell University Medical College, New York City
THIRTEEN FIGURES

The minute structure of true reticular tissue (reticulum, Mall)
has long been a matter of more or less controversy. Kolliker and
his followers regarded it as composed of branching, anastomosing
cells, while Bizzerzo, recognizing and laying more stress upon
its fibrous character considered it composed of bundles of fine
fibers to which the fixed connective tissue cells are closely applied.
As Mall (96) has pointed out it makes but little difference
whether the fibers are within or without the cells provided we
understand what is the precise relation.

A few fundamental facts of structure may be taken for granted.
That reticulum contains both fibers and fixed connective tissue
cells is obvious. That it is more or less infiltrated by leucocytes
is well known. That its anastomosing elements are frequently
continuous with bundles of white fibers may be readily observed
in any section of lymphoid tissue. , The points of divergence ap-
pear when one attempts to determine the relation: (1) of fixed
connective tissue cells to reticular fibers (‘Gitterfasern’), (2) of
reticular fibers to elastic fibers, (3) of reticular fibers to the white
fibers of collaginous tissue, (4) of ‘fixed’ to ‘wandering’ cells.
The present paper is concerned with the attempt to throw some
light upon the basic problems related to the first three of these
questions.

277

278 J. S. FERGUSON

The early studies of Kélliker, Ranvier and others, were largely
conducted upon teased and unstained reticulum or upon lymphoid
tissue from which the lymphocytes had been washed out by vari-
ous methods. They were followed by the employment of the
more recent dye reactions by which the recognition of the fibrillar
character of the tissue is rendered somewhat more apparent. It
was not until the more exact methods of chemistry and micro-
chemistry were applied by Siegfried, Mall and others that a fairly
clear perception of the exact structural relation of the reticular
tissue began to be apparent. The introduction of new methods
often renders plain certain hitherto obscure facts. This is espe-
cially true as a result of the silver impregnation methods of Biel-
schowsky (’04) when applied to various connective tissues. Even
the finer fibers, which are more or less obscure after preparation
with other methods, stand out clearly in these preparations and
one is thus enabled to draw sharper distinctions than is otherwise
possible. It is with the application of this method, and its modi-
fication by Maresh (’05), that my observations were largely made;
the results have been confirmed by comparison with consecutive
serial sections stained by well known methods, chiefly depending
on haematoxylin and eosin, Mallory’s fibroglia stain, and the
combination of haematoxylin with the Weigert and Van Giesen
stains described in my Textbook of Histology (05). ‘The tissues
studied have been lymphatic glands, spleen, tonsil, thymus, the
lymphoid tissues of the digestive and respiratory tracts, the skin
and various other tissues being used for comparison. The mate-
rial was obtained from man, pig, dog, cat, rabbit, ox, sheep, calf
and fish. It was fixed by the various methods in common use and
was both mature and embryonal.

The method of impregnation which I have followed has been
a variation of the rapid modification described by Maresh (’05).
With individual exceptions I have gotten uniformly good results
after all the methods of fixation used. The method was applied
as follows:

1. Sections cut in paraffin were fixed on the slide and placed
12 to 24 hours in a 2 per cent solution of silver nitrate.

2. Transfer for 15 to 30 minutes to freshly prepared alkaline
silver solution (20 ec. of 2 per cent silver nitrate to which are added

RETICULAR AND OTHER CONNECTIVE TISSUES 279

three drops of 40 per cent caustic soda and the precipitate redis-
solved by adding ammonia drop by drop while stirring).

3. Rinse quickly in distilled water and place in 20 per cent for-
malin for three minutes or till the sections are black.

4, Wash in distilled water and place for ten minutes in an acid
gold-bath (10 cc. distilled water to which are added 2 to 3 drops
of 1 per cent gold chlorid and 2 to 3 drops of glacial acetic acid).

5. Immerse in 5 per cent hyposulfite of soda 3-1 minute to re-
move all unreduced silver.

6. Wash in distilled water, dehydrate, clear in xylol and mount
in balsam.

Bielschowsky advised leaving tissues in the 20 per cent formalin
for 12 to 24 hours but as Maresh has shown this seems to be un-
necessarily long, at least when sections are used. To still further
shorten the process Woglom (’09, ’10) has advised, for the purpose
of preventing shrinkage of the tissue, that the initial immersion
in 2 per cent silver nitrate solution need not exceed 5 minutes.
I have, however, found this time entirely too short in many cases
and its use led to much confusion in the interpretation of my early
results. In sections insufficiently impregnated the contrast
between collaginous and reticular fibers was not sharp, either the
reticulum taking a brown instead of a proper black color in lightly
toned preparations or if the toning was intensified many of the
collaginous fibers became a greyish black instead of the proper
golden brown. Moreover, I did not find troublesome shrinkage

of tissue in well fixed preparations. I would therefore advise
a strict adherence to the 12—24 hour period of immersion recom-
mended by Bielschowsky and Pollock (’04), Levi (06) and others,
rather than the shorter period advocated by Woglom and, with
reservations, by Maresh (’05).

In the lymphoid tissues the impregnation brings out most
distinctly the reticular fibers; they take on a deep opaque black
and stand out prominently against the golden brown of the collag-
inous fibers. The method has already been applied to connective
tissues and the assumption of a more or less specific staining
property for reticulum in the liver, lymphatic glands, tonsil,
spleen, ovary, and pleura has been casually recorded by Maresh
(05), Ciaecio (07), Alagna (’08), Balabio (’08), Cesa-Bianchi

280 J. S. FERGUSON

(08), and Favaro (’09). Realizing the uncertainty which
attends the use of various silver methods one readily appreciates
the necessity for careful study of the effects of the method upon the
various tissue elements. Of the primary tissues epithelium and
other cells are scarcely if at all colored or have a faint brownish
tint; red blood cells darken readily and are either opaque black
or a deep brown according to the depth of the impregnation and
the duration of the toning bath; blood serum and intercellular
cement substance blacken, the latter appearing very granular;
all nuclei are an intense black, the silver reacting specially to the
chromatic portions, viz., nuclear wall, chromatin net and karyo-
somes; the axis cylinders of nerves are somewhat blackened,
though with the method employed the neuraxes are not nearly
so opaque as the fibers of reticular tissue. Muscle fibers blacken
irregularly depending on the depth of impregnation and they
show something of their fibrillar structure; the cross striations
and, in smooth muscle, the myofibrillae and intercellular bridges
appear beautifully shown in certain instances but it is possible
with care to have the muscle almost colorless and the reticular
fibers an opaque black. The silver apparently adsorbs somewhat
to the surface of muscle cells and elastic fibers and thus frequently
fills the interstices between fibers, forming an apparent inter-
fibrillar network in smooth muscle, in epithelium and in dense
elastic tissue, e.g., ligamentum nuchae. It is possibly this which
accounts for the apparent blackening of intercellular substance.
Both because of their reactions and because of their character-
istic differences of structure one has little difficulty in differenti-
ating these tissues after impregnation and distinguishing them
from the various types of connective tissue fibers.

Before we can regard the method of Bielschowsky, applied to
tissues outside of the nervous system, as a specific stain for reticu-
lum, it is necessary to examine more carefully than has been done,
into the reaction of connective tissue fibers to the silver impregna-
tion and the differentiation, in sections prepared by this method,
of the collaginous, elastic, fibroglia and reticular fibers. Of the
fibrous tissues cartilage, bone, and dentine may be set aside be-
cause of their characteristic structure, obvious at a glance, though

RETICULAR AND OTHER CONNECTIVE TISSUES 281

one sometimes encounters difficulties in the transition from the
fibrous perichondrium to the cartilage matrix.

The distribution of the fibers blackened by silver is so extensive
that one is tempted to question the selective action of the method.
They are encountered in all the lymphoid organs, in the mucosa
of the digestive tract, In and about the walls of the lymphatics
and blood-vessels, in the framework of all the secreting glands,
e.g., liver, salivary glands, pancreas, mucous glands of the respir-
atory and digestive tracts, in the kidneys, ovary, uterine wall,
testis, prostate, sweat glands, in the corium of the skin, in the
tunicae propriae of the respiratory and digestive apparatus,
about the glands of the gastro-intestinal mucosa, and to a limited
extent among the fibers of areolar and collaginous tissue wherever
it is found. Many of the basement membranes consist largely
of these argentiferous fibers.

If one is careful not to overtone the specimens the results in
mature tissues are fairly constant; in such preparations (with a
few reservations) the method appears quite definitely selective
for the blackened fibers of reticular tissue (‘‘reticulum.’’ Mall),
the elastic and fibroglia fibers remain colorless and the collaginous
fibers assume a brownish tint.

This result was arrived at by a careful comparison of the effect
of this and other stains upon the several tissues in locations where
each is known to occur. The conclusions are based on the fol-
lowing observations.

A. ELASTIC FIBERS

Sections of the ligamentum nuchae after impregnation show the
elastic fibers absolutely colorless and outlined by an intense black
fibrous mass which occupies nearly the entire non-vascular area
between the elastic fibers, and which at the borders of the elastic
bundles shades into the golden brown of the collaginous fibers
forming the coarse bands of the framework (fig. 1). In the con-
trols the elastic fibers show the characteristic staining reaction
with hematein and eosin and with Van Giesen’s stain, and the
intervening tissue is colored red by eosin and by acid fuchsin.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 12, NO. 3

IRD J. S. FERGUSON

Fig. 1 Transection of the ligamentum nuchae of an ox. Bielschowsky stain.
The elastic fibers are colorless, the intervening tissue opaque. Camera lucida;
Tae, Ie Oils Ge

Fig. 2 A pulmonary artery from the human lung showing the colorless internal
and external elastic membranes invested by blackened reticulum. Bielschowsky
stain. Camera lucida; occ. 1, obj. ¢-

Fig. 3 From a primary bronchus of man. Basement membrane (bm) is a dense
opaque black, its fibers so closely packed as to be indistinguishable. The elastic
fibers (ef) are colorless and are invested by a black reticulum (r). Bielschowsky
stain. Drawn with the Edinger projection apparatus, X 250.

In the fenestrated coats of the arteries one sees in the larger
arteries of the lungs, stomach, lymphatic glands and many other
organs vessels showing in the controls the typical elastic membrane
in the tunica intima completely encircling the vessels in transec-
tions. The impregnated specimens, when the same vessel is
examined in adjacent slides in the series, show the internal elastic
membrane colorless, the elastic coat in the larger vessels being in-
vested on either surface with a close net of black reticular fibers
(fig. 2). This investment of the elastic tissue by reticular fibers
is readily observed and is most remarkable. Similar, though less
numerous fibers are seen investing the elastic tissue in the inter-
muscular spaces of the tunica media, and in the tunica adventitia.

RETICULAR AND OTHER CONNECTIVE TISSUES 283

In the smaller arteries and in the small and medium veins the coat
of Henle is so thin, and often incomplete, that it is more difficult
to determine that the elastic fibers are colorless as distinguished
from the blackened reticulum but in view of the constant and
obvious condition in the larger vessels one is warranted in assum-
ing that the elastic fibers in the smaller vessels, as in the larger,
are colorless and that it is the reticulum, when present, which
blackens. The intimate clothing of elastic fibers by reticulum,
readily observed in the larger vessels, accounts for the occasional
appearance of blackened fibers in the position of Henle’s coat in
vessels so small as to possess only an incomplete internal elastic
membrane.

In the basement membranes of the bronchi one finds only
argentiferous reticular fibers. In the larger bronchi the basement
membrane is specially distinct and consists of a dense, closely
packed mesh of blackened reticular fibers (fig. 3), forming a com-
plete membranous investment continuous with the reticular fibers
of the tunica propria and supporting the epithelium. With the
Weigert-elastic picro-fuchsin stain the argentiferous fibers take
a red color.

In the tunica propria of the trachea and bronchi are large bun-
dles of longitudinal elastic fibers. These fibers remain colorless
in the Bielschowsky sections even when the stain has been made
so intense as to darken to a considerable extent the collagimous
fibers and the muscles. One finds each elastic fiber invested by a
distinct coat of blackened reticular fibers forming an intricate
net. If one selects a known and readily recognized point for
study, consecutive sections stained by different methods show
the broad lines of elastic fibers, which in the Bielschowsky sec-
tions are colorless, to be flanked on every surface by a blackened
reticulum, but clothed in the Weigert-elastic picro-fuchsin section
by fuchsin stained fibers. With haematoxylin and eosin the whole
breadth of the basement membrane and both elastic and argentif-
erous fibers in the tunica propria take the characteristic eosin
tint, and reticular and elastic fibrils are almost indistinguishable.

Thus wherever the recognition of unquestionable elastic fibers
can be made with certainty they are found uncolored by the silver,

IR4 J. S. FERGUSON

while giving characteristic reactions to other stains. It is only in
those locations where identification of elastic fibers is question-
able that one is inclined to suggest their identity with the black-
ened fibrils, but even then one sees indications of noticeable
difference. If a bit of areolar tissue is carefully examined in the
wall of the digestive tract, the skin, the peritoneum or elsewhere,
and sections of the tissue are also stained by the selective elastic
tissue stains, Weigert’s, or Unna’s, one observes on comparison a
difference in the number and arrangement of fibers selected by the
compared methods. The orcein and Weigert sections will com-
pare very closely. The Bielschowsky sections of the same series
frequently showmany more fibers of the blackened type; moreover
the blackened or reticular fibers are usually more wavy and of
irregular distribution, often having a typical spiral appearance
as compared with the relatively straight elastic fibers. This is
well shown in sections of collapsed or undistended lung in which
the elastic fibers of the alveolar walls and bronchioles are straight
while the reticular fibers, from the extreme contraction of the
organ, are thrown into a remarkably intricate network of wavy
and twisted fibrils, equally as distinct and more abundant than the
elastic.

These findings are in confirmation of the views already expressed
by Woglom (710) and others and appear to prove conclusively the
non-identity of elastic fibers with those fibers (reticulum) which
blacken in these preparations. This view is in accord with that
expressed by Mall (02), who as a result of his comparison of the
tissues by chemical methods likewise demonstrated the non-
identity of elastic fibers and reticulum, but his studies of mesen-
chymal tissues showing embryonic stages of the connective tissue,
resulted in pictures delineating the first appearance of elastic
fibrils which simulate those which I have obtained in similar tis-
sues by the method of impregnation (fig. 4). 1 shall consider this
phase of the subject in a later paper. At this time it is sufficient
to say that I consider the fibers referred to to be collaginous in
type. One must therefore finally emphasize the fact that in well
recognized portions of mature tissues elastic fibers in the silvered
preparations remain entirely colorless while reticular fibers

RETICULAR AND OTHER CONNECTIVE TISSUES 285

he } °

Hone oo “.

\\) e

hide! 5

Fig. 4 From the subectodermal mesenchyme (corium) of a fetal pig of about
80 mm. neck-breech length, showing blackened fibrils (F) bearing intimate rela-
tions to the mesenchymal cells. Bielschowsky stain. Camera lucida; occ. 1,
obj. ;'5 hom. im.

Fig. 5 Reticular and collaginous tissue at the periphery of a lymphatic gland.
Observe the sharp outlines of the fine black reticular fibrils (7) which intermingle
with the bundles of collaginous fibers (cf) in a trabeculum. Bielschowsky stain.
Camera lucida; oce. 1, obj. %.

Fig. 6 The figure exhibits the relation between the collaginous (cf) and reticular
(r) fibers in the region adjoining a nodule of a patch of Peyer in the small intestine
of man. Note the interlacing of fibers at the border of the lymphoid tissue.
Bielschowsky stain. Camera lucida; oce. 1, obj. §

6°

blacken; one is justified in assuming that this reaction to the
Bielschowsky method, with reasonable care, is constant.

B. COLLAGINOUS FIBERS

That the typical reticular fibers of lymphoid tissue and the
typical collaginous fibers of dense fibrous tissue take on differ-
ent colors after the silver impregnation has been generally
recognized, at least by the Italian writers, Levi (’06), Ciaccio

286 J. S. FERGUSON

(07), Cesa-Bianchi (08), Balabio (08), Alagno (08). The retic-
ular fibers assume a dense opaque black while the collaginous
fibers take on a golden brown when well differentiated. Yet
if one examines carefully those points at which the two tissues
blend one encounters much difficulty in determining whether the
black color of the coarser collaginous bundles is due to the opacity
of the brown bundles—which are of considerable size and thick-
ness and often of great density—or to the presence within the
coarse collaginous bundles of finer, blackened, reticular fibers.
In some locations the latter relation is apparent. For example,
in the perifollicular plexus about the lymphatic follicles, described
by Ciaecio (07), one finds the characteristic lozenge shaped
meshes of the ‘reticulum’ extending into the adjacent collaginous
tissue of the trabecula in lymphoid organs or of the tunica pro-
pria and submucosa in the digestive tract, but there the reticular
fibers are nearly always clear and sharp among the collaginous
fibers of the smaller fibrous bundles (figs. 5, 6, 7, and 13). As the
bundles increase in size, however, the difficulty of distinguishing
the exact outlines of the two types of fibers increases.

Another difficulty in the way of exact and positive differentia-
tion is the variable result of silver impregnations. With vary-
ing degrees of impregnation, reduction, and toning the collaginous
fibers may lose their typical golden brown and acquire an increas-
ingly opaque condition. This is specially prone to occur if the
sections are overtoned in the gold chlorid bath. One halts, there-
fore, between the idea of similarity if not positive identity of col-
laginous fibers and ‘‘reticulum”’ and the opinion of Mall (’01)
which regards reticulum as an independent tissue, distinctly dif-
ferentiated from the collaginous by its somewhat different chem-
ical reactions, a view not fully accepted by Studnicka (’03) nor
yet generally adopted by German authors (Furbringer, 09). Yet
if one uses care with the silver process one can obtain from nearly
all tissues quite distinctive preparations. Thus in the lung the
fibrous tissue of the pleura, as shown by Favaro (09), as well
as that of the “‘interlobular septa” appears to be formed by golden
brown fibers arranged in bundles having the characteristic wavy
course together with but few intermingled black reticular fibers,

RETICULAR AND OTHER CONNECTIVE TISSUES 287

whereas the reticulum in the walls of the alveoli and smaller bron-
chi, though often composed of coarse typically spiral fibrils, forms
an interlacing mass of discreet fibers, or fiber bundles, among
which a limited proportion of finer bundles of brownish collag-
inous fibers may be recognized. In the vascular trabecula of the
spleen (fig. 7) the collaginous fibers of the blood-vessels acquire
a typical brown while the close network of reticular fibers take on
an intense black and have a characteristic, either somewhat
regularly spiral, or a reticular course, very different from the
irregularly wavy collaginous fibers.

Fig..7 A vascular trabeculum of a child’s spleen. The blackened fibers of
reticulum (7) show clearly in contrast to the collaginous fibers (cf) which in the sec-
tion are a golden brown. The reticulum surrounds the vessels and is continuous
with that of the splenic pulp. Bielschowsky stain. Drawn with Edinger pro-
jection apparatus, & 255.

In the trabecula of the lymphatic glands the distribution is not
so apparent, the collaginous and the reticular fibers pursuing some-
what similar courses, though the latter are apt to be more dis-
tinctly spirillar. From careful examination I am led to believe
that the relation simulates, in reverse, that already described (see
fig. 2) for the elastic fibers, in that it would appear with consider-
able certainty in many places that the black reticular fibers are
invested or enveloped by a sleeve or coat of collaginous fibrils,
so that the latter fibrils consequently assume a spiral course cor-
responding closely to that of the reticular fibrils. Indications of
a similar investment of the reticular fibrils can be found wherever
reticulum occurs, but it is not always possible to distinguish
with certainty between the collaginous fibers and the protoplasm
of mesenchymal or fixed connective tissue cells.

288 J. S. FERGUSON

Again in the fibrous perichondrium of hyaline cartilage, as
Studnicka (’06) has pointed out, there is a considerable layer of
blackened fibers marking the border of the cartilage and in the
younger types extending into its matrix; the outer layers of the
perichondrium are clearly, however, collaginous tissue, and in
my preparations present the characteristic golden brown color,
sharply distinguished from the intense black of the argentiferous
fibers. The matrix of the cartilage in the same sections retains
a brownish tint except in the younger specimens and at the margins
of the cartilaginous plates in the more mature cases. The black-
ening of the innermost fibers of the perichondrium which mark
the ‘‘growing surface”’ of the cartilage may be explained by the
increased affinity for silver shown by the fibers of young connec-
tive tissue as compared with the mature, a relation which I am
not ready to discuss further at this time.

The remarkable differences in reaction to the impregnation in
many of the mature tissues, especially such as contain typical
reticulum, would tend to refute the German idea of the identity of
collaginous and reticular tissue and to confirm the opinion of Mall
that reticular tissue or “reticulum” is a distinct entity, though this
latter contention cannot yet be established from the standpoint
of the method here used until it is viewed in the light of the his-
togenesis of the connective tissues, for there the sharp lines of
demarcation diminish even to the vanishing point.

Such characteristic differences between reticulum and collag-
inous fibers as may be observed at almost any point in thin sec-
tions of the lymphoid tissues impregnated by silver leave little
to be desired in the way of morphological differentiation of these
two types of fibers. Such areas are well and accurately shown in
fig. 5, from the lymphatic gland of man, and fig. 6 from the margin
of a Peyer’s patch in the human intestine. One feels, therefore,
that the separate and distinct character of collaginous and retic-
ular fibrils in the mature tissues as shown by silver impregnations,
fortified as it is by the chemical differences demonstrated by Sieg-
fried and Mall, the one, collaginous, yielding gelatin, the other
yielding a “‘reticulin” presenting different chemical reactions,
forms at least a satisfactory working basis for the further study

RETICULAR AND OTHER CONNECTIVE TISSUES 289

of the distribution of these fibers as shown by the Bielschowsky
method, a work already begun by Studni¢ka, Ciaccio, Balabio,
Alagna, Favaro, Maresh, Cesa-Bianchi and others.

C. RETICULUM

The careful observation of Bielschowsky preparations also
yields valuable data as to the finer structure of reticular tissue
and the relation of its fibrils to the ‘‘ fixed” connective tissue cells.

The coarser fibers of ‘reticulum’ may be readily seen and, where
such fibers come into relation with the ‘‘knots” of the reticular
net, one can observe these fibrils breaking up into a plexus within,
or about, the cells as pointed out by Balabio (08). Somewhat

Fig. 8 From a lymphatic gland of man showing the relation of the blackened
fibers of reticulum to the branching protoplasm of the fixed connective tissue cells.
The small black nuclei are those of lymphocytes. PR, perifollicular reticulum.
Bielschowsky stain, after stained with acid fuchsin. Camera lucida; occ. 1, obj.
iy hom. im.

of this arrangement is indicated in fig. 8, though in other places
the fikers appear to enter the cell and end either abruptly or,
more frequently, pass through the cell in close proximity if not in
contact with its nucleus. The appearance of abrupt ending
might if only occasionally observed, be due to the passage of fibers
out of the plane of the section, but it occurs far too often so that
this certainly is not always the case. The finer fibrils, as well as
many of the coarser ones, appear as single fibrils though because
of the complete opacity of the impregnation one cannot say that
this is actually the case. Certainly the larger fibers distinctly
show indications of fibrillation.

290 J. S. FERGUSON

This leads to the question of the relation of reticular fibrils
to the ‘‘fixed’”’ connective tissue cells. Are the fibers contained
within the cells or are they only in surface contact? It seems to
me that Mall has given us the key to the situation with his theory
of exoplasmic deposit of the fibrils with constant recession of the
endoplasm during development. If one regards reticulum as an
immature or least differentiated type of connective tissue it 1s plain
to see that the fibers must readily ie now within the cell or endo-
plasm, and now without the cell, where they are left “high and dry,”
as it were, by the complete recession of the endoplasm which leaves
in mature collaginous tissue only the nucleated cellular remnants.
Since certain fibers, or portions of fibers, would thus he without
the anastomosing syncytial mass of endoplasm while certain
others would le quite as plainly within it we have here a possible
harmonization of the otherwise conflicting theories of Kolliker
and Bizzozero. The facts of the case as I observe them in silver
impregnated sections of embryonal as well as mature tissues ap-
pear to coincide with this hypothesis.

Ciaccio (07) attacked this problem casually in connection with
his study of the distribution of reticular tissue in the lymphoid
follicles of lymphatic glands and observed a relation of contiguity
of fibers and cells, the two being independent. Thus, he says,
“le fibrille alla loro volta si diramano in tutti 1 sense e si montrano
independent dalle cellule.”

Balabio (O08), cognizant of the work of Ciaccio, approaches the
problem circumspectly, and describes the fibers as “‘superimposed”’
upon the cells forming a characteristic close and delicate peri-
cellular plexus. He observed that the cellular prolongations “‘in-
tertwine among” the fibrils but he was not able to determine “with
certainty’ whether they were superimposed or whether they ‘‘an-
astomosed in the form of a sort of continuous cellular net.” He
“limits himself,” as he says “to emphasize the fact without pro-
nouncing upon the existence or non-existence of true cellular
anastomoses.’’ He is inclined to confirm the theory of Bizzozero
for he in one place says ‘“‘Si puo confirmare conscicurezza quanto
gia Bizzozero ed altri affermarono che si tratta di rapporti di sola
contiguita.”’

RETICULAR AND OTHER CONNECTIVE TISSUES 291

If one assumes that to disprove the theory of Bizzozero one
must find the fibrils at all times outside the anastomosing cells,
never within, then proof is not forthcoming. On the other hand
proof is also lacking if to demonstrate the theory of Kolliker
fibers must always be found within the cellular syncytium. But
viewing the tissue in the light of its histogenesis, one need not, as
pointed out above, be thus limited in either case, for the fibers of
reticulum may, according to this interpretation, come to lie now
within, now without the syncytial endoplasm. The examination
of such appearances as those shown in any of the figs. 8 to 11,
which are accurately drawn with high magnification, or yet more

Fig. 9 Reticulum and cells as seen in a thin section through the pulp of the
human spleen. No collaginous fibers have been included. Fibers and nuclei are
black, the cytoplasm is granular. The fibers are surrounded by a halo of cyto-
plasm especially distinct wherever their cut ends are directed toward the eye
of the observer. Ret, reticlular fibers; fc, cytoplasm; L, lymphocyte. Biels-
chowsky stain. Camera lucida; occ. 1, obj. ;’y hom im,

truly if one studies the actual preparations, must convince one at
a glance that in mature lymphoid tissue the fibrils of the reticu-
lum are not entirely contained within the fixed cells. The burden
of proof lies on the other side.

292 J. S: FERGUSON

The accurate and sharp delineation possible under high magni-
fication between the opaque black fibrils and the light brown
protoplasm of the cells presents appearances in thin sections which
seem to me to show unmistakably that some portions of the fi-
brils are certainly contained within the cytoplasm of fixed connec-
tive tissue cells. I do not find any such condition in relation to
the lymphocytes which are so numerous in the same vicinity.
In fig. 10 the fibrils a are, in the case of the lower cell at least, cer-
tainly outside the cell at one point, viz., where it ends by passing
out of the plane of the section. But at the point 6 each fiber
makes a distinct loop which can be followed by change of focus.
The granular cytoplasm forms a continuous mass but in the mid-
portion of the loop it can be distinctly seen at a level above that of
the fiber; while at the ends of the loop, in fact at all the solid black
portions of the fiber the cytoplasm is distinctly below the fiber.
It would appear obvious that each fiber has penetrated the cell
and must, therefore, during its passage have been found within
the cytoplasm. Fig. 10 was drawn from a section of the spleen,
but in fig. 11, which is from a lymphatic gland and in which a
again marks the portion of the fiber above, and 6 that below the
cytoplasm, the same condition holds. Such places are extremely
abundant, and in thin sections of all the lymphoid tissues ex-
amined they can be found with ease, often several in a single
field. Again in transections of the coarser fibers, or in oblique
sections, the fibers are often seen surrounded on all sides by a
light brown halo of cytoplasm. Such appearances are indicated
by fig. 9, though it is difficult to depict them accurately even with
the aid of the camera lucida because of the extreme fineness of the
fibers and the very thin cytoplasmic coat (represented by the
stipling) by which they are surrounded. The cut ends of most of
the transversely and obliquely cut fibers show the halo of cyto-
plasm in the actual sections.

The above observations appear to show convincingly that, at
least, at times the fibrils lie distinctly within the cells. That they
may be so found, as also without the cells, is in harmony with
Mall’s suggestion as to the ontogenetic relationship of ‘“reticu-
lum’ and other connective tissues since he supposes this tissue

RETICULAR AND OTHER CONNECTIVE TISSUES 293

to represent a less mature type than the collaginous, one in which
the primitive relations of endoplasm and exoplasm still persist to
a considerable extent. That this is the case is, perhaps, indicated
by the fact that in developing mesenchymal tissue one finds
fibrils, bearing similar relations to the endoplasm and exoplasm
of the connective tissue syncytium, and which like reticulum
blacken with the silver impregnation (fig. 4).

Fig. 10 Accurately drawn from a section of the spleen of man, showing the
actual course of fibrils of blackened reticulum through the cytoplasm of fixed
connective tissue cells. The parts a of the fibrils end by making a sharp turn
which passes out of the plane of the section. The loops formed at b are shaded
light, and in the section they lie below the level of the cytoplasm as readily demon-
strated by change of focus. The black portions, a, are above the level of the eyto-
plasm. Bielschowsky stain. Camera lucida; occ. 1, obj. y'5 hom. im.

Fig. 11 Areas similar to those shown in fig. 10, but drawn from a section of a
lymphatic gland of man. Similar appearances were very numerous in this section.
a and 0b, as in the preceding figure; cy, cytoplasm; f cut ends of fibrils; L, lympho-
cytes; Nu, nucleus of a fixed connective tissue cell; at the top of the figure a
fibril forms a U-shaped loop which passes through the cytoplasm of a “‘fixed’’ cell,
entering from below and coming out above. Two similar fibers are also shown.
Bielschowsky stain, after stained with acid fuchsin. Camera lucida; oce. 1, obj.
x hom. im.

Yet bearing in mind that we are dealing with a method of
impregnation only, and are subject to all the limitations of such
methods, one is not fully warranted in drawing inferences of chem-
ical similarity between the mesenchymal and the reticular fibrils.

294 J. S. FERGUSON
D. FIBROGLIA

The occurrence of fibrils blackening with silver in the mesen-
chymal cells suggests a possible identity with the fibroglia fibrils
of Mallory (08, ’04) for such fibrils were found by that observer
to be abundant in developing connective tissue. In order to
accurately compare the fibrils shown by these two special methods
one must first consider the mature tissues, only thereafter the
developing tissues.

In mature tissues Mallory states that fibrogha fibrils ‘‘are not
very common in normal tissues except possibly in one situation
and have to be hunted for with an oil immersion lens.’ This is
certainly not the case with the argentiferous fibers which occur
abundantly in a great variety of places among normal tissues
and which are of sufficient size to be seen as networks among

‘

Fig. 12 The basket-cells of a coil gland of the human finger-tip, darkened by
haematoxylin. Haidenhain’s iron-haematoxylin. Camera lucida; occ. 1, obj.
iy hom. im,

the other fibers with very low magnification. Again the
staining reactions of the two sets of fibrils are different. Mal-
lory describes the basement membranes as the ‘‘one situation”
where fibroglia fibrils are common in normal tissues, and the sub-
epithelial basket-cells of the sweat glands—regarded by Benda
(°93, ’94) as muscle cells—as the place where the largest fibrogha
fibers occur. As these last fibers can be easily located they form
a definite unit for comparison. With Mallory’s stain they are
red; with iron haematoxylin they blacken when the stain is not
too much extracted (fig. 12). Both of these reactions are char-
acteristic for fibroglia, and, as MeGill (08) has shown, they are
also characteristic for myoglia fibrils. But with silver impregna-
tion these fibers are not in the least blackened, nor is the thin

RETICULAR AND OTHER CONNECTIVE TISSUES 295

layer of collaginous tissue upon which they rest. It would there-
fore appear that the basement membrane of the sweat glands,
unlike most other basement membranes, contains no reticulum
but is formed by collaginous fibers together with the peculiar
basket-cells, be they fibroglia or muscle. If one compares in the
same way the reticulum of lymphoid tissues one arrives at similar
conclusions as to the non-identity of ‘reticulum’ (viz. those fibers
which blacken with the Bielschowsky method) and _fibroglia.
It would therefore appear that in the mature tissues there is no
identity between fibrogla and reticulum nor for the same reasons
can there be between fibroglia and the fibers which blacken with
Bielschowsky’s stain. These last are identical with certain fibers
which are colored blue by Mallory’s stain.

SUMMARY

Briefly summing up we find that the Bielschowsky stain applied
to the connective tissues of mature individuals exerts a selective

Fig. 13 A small lymphatic nodule from the submucosa of the human esophagus,
showing the ‘perifollicular plexus’ of Ciaccio sharply defined, but with reticular
fibers intertwining with the collaginous fibers.

The collaginous tissue is drawn free-hand, the reticulum by camera lucida;
occ. 1, obj. %.

action, blackening certain fibers which are certainly not identical
with either elastic or fibroglia fibers, which in many cases certainly
are identical with the fibers of reticulum, and which in some cases
show a certain tendency suggesting possible transitions between
reticular and collaginous fibers. The typical collaginous fibers do

296 J. S. FERGUSON

not blacken, but take on a characteristic golden brown color;
nevertheless, in certain locations and under certain conditions
some fibers which we have been accustomed to regard as collag-
inous, e.g., within dense connective tissue bundles or in embry-
onic mesencyhme, do blacken somewhat, though never so typically
nor with such clear and sharp definition as do the fibers of “‘reticu-
lum”? in mature tissues. The further elucidation of this atypical
reaction of the collaginous fibers must be sought in the histogen-
esis of the connective tissues. For the present we may safely
consider the black reaction of fibers of mature connective tissue
to the Bielschowsky stain to be distinctive of “‘reticulum”’ in all
satisfactory preparations, viz. those in which the collaginous
fibers assume a golden brown tint.

When such tissues as nerve, muscle and embryonic mesenchyme
are excluded the Bielschowsky method serves as a well-nigh
specific stain for the reticulum of Mall.

BIBLIOGRAPHY

Auaena, G. 1908 Anat. Anz., Bd. 32, p. 178.
BauaBio, R. 1908 Anat. Anz., Bd. 33, p. 135.
Benpa_ 1893-1894 Dermatol. Zeitschr., Bd. I, p. 94, (Quoted by Mallory).
BrELScCHOWSKY, M. aNnp Poutuack, B. 1904 Neurol. Centralbl., vol. 23, p. 387.
Crsa-Brancu1, D: 1908 Anat. Anz., Bd. 32, p. 41.
1908 Internat. Monatschr. f. Anat. u. Physiol., Bd. 25, p. 1.
Craccio, C. 1907 Anat. Anz., Bd. 31, p. 594.
Favaro, G. 1909 Internat. Monatschr. f. Anat. u. Physiol., Bd. 26, p. 301.
Frracuson, J.S. 1905 Normal histology and microscopical anatomy, New York
and London, pp. 666-7.
FURBRINGER, M. 1909 Gegenbaur, Lehrb. d. Anat., 8 auf., Bd. 1.
Levi, G. 1907 Monitore zool. ital., 18, 290.
McGitu, C. 1908 Internat. Monatschr. f. Anat. u. Physiol., Bd. 25, 90.
Mau, F. 1896 J. Hop. Hosp. Rep., vol. 1, p. 171
1902 Amer. Jour. Anat., vol..1, p.’329.
Mautory, F. 1903-1904 Jour. Med. Research, vol. x, p. 334.
Marescu, R. 1905 Centralbl. f. allg. Pathol., Bd. 14, 641.
Stecrriep, M. 1892 Habilitationsschrift, Leipzig, (quoted by Mall).
1902 J. of Physiol., 28, 319.
Stupnicka 1903 Anat. Hefte, Bd. 21, 279.
1906 Zeitschr. f. wis. Mik., Bd. 23, 414.
Woctom, W.H. 1909-10 Proc. N. Y. Path. Soc., vol. 9, p. 146.

{Reprinted from BroLocicat BuLLETIN, Vol. XXI., No. 4, September, 1911.

’

A PRELIMINARY NOTE ON THE RELATION OF NOR-
MAL LIVING CELLS TO THE EXISTING THEORIES OF
THE HISTOGENESIS OF CONNECTIVE TISSUE.

JEREMIAH S. FERGUSON, M.S., M.D.,

ASSISTANT PROFESSOR OF HISTOLOGY, CORNELL UNIVERSITY
MEDICAL COLLEGE, NEW YorkK CIty.

The connective issue of the adult is in the embryo derived
from the mesenchyme. The same is true of certain other
tissues, endothelium, muscle, and cartilage, bone and dentine,
if these last be not included within the scope of the term con-
nective tissue. It is in this narrower sense that the term con-
nective tissue is herein used.

In considering the origin of connective tissue one has to
take into account two distinct elements, cells and fibers. Of
the cells there are at various stages in the embryonic mesenchyme
three distinct types: (a) the wandering cells, viz., leucocytes,
which are more directly concerned with the processes of he-
matopoiesis; (b) those cells which give rise to the true wandering
connective tissue cells of the mature tissues, the mast cells,
plasma cells, etc., whose history is more or less closely concerned
with that of the embryonic leucocytes; (c) those typical con-
nective tissue cells which are related to the fibers and which
are commonly known as the ‘‘fixed’’ connective tissue cells.
It is without doubt only the last type which is concerned with
the origin of the connective tissue fibers, hence the present note
deals only with this type of cell.

At least two types of fibers have also to be considered as
arising in the connective tissue mesenchyme, the elastic and the
collaginous. We are here concerned only with the latter type,
for the reason that the elastic fibers arise at a later period; thus
the origin of the connective tissue fibers concerns primarily
the collaginous, or so-called “white fibers.”’ It is unnecessary
to distinguish at the early stage under consideration between
such varieties of collaginous fibers as ‘‘reticulum”’ and “ fibrog-

272

273 JEREMIAH S. FERGUSON.

lia,” for the former undoubtedly arises in exactly the same

’

manner as the ordinary ‘‘white fibers,’ and of the histogenesis
of the latter little or nothing is known if, indeed, it can be con-
sidered as an entity distinct from the other connective tissue
fibers.

The numerous theories which have been proposed to account
for the origin of the connective tissue fibers may be reduced to
three chief divisions:

1. The embryonic connective tissue or mesenchymal cells
become directly transformed by elongation into connective
tissue fibers. _

2. The fibers arise in the ground substance between the
cells either by transformation of that ground substance or as
a secretion from the adjacent cells.

3. The fibers arise within the cells either as distinct fibers
(Schwann, 1839), as granules which fuse to form fibers (Spuler,
1896; Lavini, 1909), as an epicellular protoplasmic fibrous layer
(Lwoff, 1889), or as an ectoplasm about the cell (Hansen, 1899)
or forming the syncytium (Mall, 1902).

The theory of direct transformation by elongation of cells
into fibers may well be abandoned and, except it be construed
along the lines of the various theories of intracellular origin,
it is worthy only of passing notice. Among other things the
existence of an overwhelming number of fibers relative to the
number of cells present in connective tissue argues against
direct transformation, and modern microscopical methods are
not able to detect the phases of such transformation either in
normal growing tissues or in the process of wound repair. For
several decades the results of observations have been in support
of either the intercellular or the intracellular group of theories.

To Henle (1841) we owe the theory of the intercellular
(extracellular) origin of connective tissue fibers, the fibers being
supposed to arise from the intercellular ground substance, not
directly from the cells. Latterly this theory has received but
little support unless it be from the observations of Merkel
(1895), who found that in the umbilical cord fibers appear first
to arise in locations relatively remote from the cells. Later
the cells wandered into these fibrous areas, and at a still later

HISTOGENESIS OF CONNECTIVE TISSUE. 274

period the fibers had so increased in numbers that they neces-
sarily laid in relation to the cells.

The weak point in Merkel’s deduction appears to be the dis-
regard of the locomotive properties of mesenchymal or young
connective tissue cells. If these cells, except for the incidents
of mitosis, are stationary, then Merkel’s observations would
render conclusive evidence to support the theory of the extra-
cellular origin of fibers. But that such cells possess at least a
limited power of motion simulating the amoeboid character
has long been known. An adequate theory must account for
the activity of these cells. Merkel’s theory presupposes them
to be stationary till fibers have appeared and only later to
acquire amoeboid characters.

In searching for a tissue which would offer opportunity for
the study of the activities of connective tissue cells in the living

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Fic. 1. Outlines of a stellate connective tissue cell in the caudal fin of a Fundu-
lus embryo 4.5 mm. long (one day after hatching). The figures record the moment
of completion of each drawing, the whole observation extending over a period of 24
minutes. The fish was afterward returned to water and swam for two days before
being killed for further histological use. The cell observed corresponds in appear-
ance with that shown by W. Flemming (Arch. f. Anat., 1897, Fig. 9, Plate VI.)
in the mesentery of a Salamander larva. It shows active amceboid motion and
extensive changes of form. Its locomotion was not recorded, it was quite limited.
X 800, camera lucida.

animal under normal conditions, I have found that the median
fin of young fish embryos affords a subject for study which throws
some light on Merkel’s observations.

275 JEREMIAH S. FERGUSON.

Ina Fundulus embryo of 6 mm. total length—several days after
hatching—I find in the median fin between the double layer
of cutaneous epithelium the first signs of the dermal fin-rays.
Blood-vessels have not entered, and between the fin-rays are
few if any connective tissue fibers, certainly none in the distal
portion of the fin. nvading the base of the fin is a mass of mes-
enchymal cells, mostly of the round cell type. Scattered through
the fin are, here and there, isolated connective tissue cells in
very limited numbers, distributed over an area representing
the proximal one half or two thirds of the fin, and forming a sort
of skirmish line, as it were, in advance of the army of round cells.
These stellate- cells can often be distinctly seen in the living
animal. Later, connective tissue cells in abundance wander into
the fin and fibers appear. But it cannot be said that the fibers
appear in advance of the cells, for the ‘skirmish line”’ precedes
the appearance of fibers, these advance cells becoming later
intermingled with those of the subsequent invasion.

The advance cells, like all other stellate connective tissue
cells in the fins of embryo fishes, I find to be actively amceboid,
capable not only of motion but to some extent of locomotion.
Since these cells are travelling through an area in which fibers
are only just beginning to appear, the location of early fibers at
points relatively distant from the cell in “‘fixed”’ tissue at once
loses its significance and thus robs the theory of the extracellular
origin of fibers of one of its strongest supports. My observations
may be readily verified on any living, free-swimming Fundulus
embryo within the first week after hatching.

Connective tissue fibers may be supposed to arise as a product
of secretion of the mesenchymal cells, the extracellular deposition
of fibers thus occurring under the influence of the cells. This
is but a slight deviation from the strict extracellular origin pre-
supposed by the theory of Henle and Kolliker. Such secretion
must appear either in fluid form or as granules. In either case
it is difficult to conceive why the secretion should take on a
linear form if the cells remain stationary, and still more difficult
if the cells are in motion, unless that motion be extremely slow
and in one direction only.

By observation of the fins of embryo fish, chiefly Fundulus,
I have been able to observe that the spindle cells of embryonic

HISTOGENESIS OF CONNECTIVE TISSUE. 276

connective tissue possess locomotion and move largely in one
direction, but their motion and locomotion is extremely active,
and so far as I can observe in the living animal they leave behind
no trail of secretion, certainly no observable granules: still more
important, the fibers appear to have been formed in great numbers
prior to the appearance of the type of spindle cells in any con-
siderable proportion relative to the other types of cells, round
and stellate, already present in the primitive connective tissue.
Hence I cannot conceive of the spindle cells as producing fibers
by direct secretion.

But it is at the time of the predominance of stellate cells that
the fibers make their first appearance in the fish’s fin, as is
likewise the case in the tissues studied by other observers.
These stellate cells rapidly change their form, throwing out and
retracting their processes in rapid succession, as can be seen in
any living, free-swimming Fundulus embryo under 25 mm. total
length. Fig. 1 shows the outline of such an early connective
tissue cell in the caudal fin, its changes of form at intervals for a
period of twenty-four minutes being accurately depicted with the
aid of the camera lucida. It is impossible to both watch and
draw all the changes in form occurring during this period, but
sufficient are shown to demonstrate very active amoeboid motion.
By observing these cells in relation to a relatively fixed object,
é. g., a chromatophore, or a joint in an adjacent fin-ray, I have
been able to detect in them considerable locomotion. I find
that, unlike the spindle cells, the stellate cells appear to travel
equally well in all directions. Neither following their locomotion
nor in trail of a retracted process do I find any indication of
secreted granules, nor of anything other than the most delicate,
preformed fibers. One would suspect that if new fibers were
being formed by a process of secretion they would appear within
twenty-four minutes after such a cell or its process had occupied
a given position in which fiber should reasonably be expected
to be formed. Though I have repeated the experiment many
times I have failed to observe such evidence of secretion, and
these results do not appear to be consistent with a theory of
direct secretion of fibers by the primitive connective tissue cells:

The intracellular theories of the origin of connective tissue
fibers may be divided into two classes, those in which the fibers

277 JEREMIAH S. FERGUSON.

are presumed to arise in the peripheral portion of the cell, as
proposed by Lwoff (1889), or in a modified peripheral portion of
the cellular or syncytial protoplasm, ectoplasm or exoplasm,
as proposed by Hansen (1899) and afterwards by Mall (1902),
and those in which the fibers are presumed to have a direct
intracellular origin as observed by Spuler (1896) and later by
Livini (1909).

The ‘‘circumcellular’’ origin of fibers proposed by Lwoff,
since it must take into account the separation of fibers from the
cells as well as their origin from intracellular granules, does not
fundamentally differ from the later ectoplasmic modifications

G Ch

le mas.

Fic. 2. A record of the locomotion of a spindle connective tissue cell, a, for
a period of 10 minutes, adjacent chromatophores bordering on the fin-rays on either
side being taken as the relatively fixed points. The cell was found to have covered
a distance of about 50u, a rate of Iu in every 12 seconds. a-—d, connective tissue
cells; c, a round cell, the others spindle shaped; Ch’, Ch’’, two chromatophores;
end, endothelium of an afferent blood-vessel.

of Hansen and Mall. These later authors, basing their inves-
tigation on ‘‘fixed”’ material, presuppose a constant but gradual
recession of the endoplasmic area with coincident changes in

HISTOGENESIS OF CONNECTIVE TISSUE. 278

the exoplasm in order to account for the transference of fibers
from a position within the cell, in the ectoplasm, to an extra-
cellular position. This hypothesis is entirely harmonious with
the appearances to be observed in ‘‘fixed’”’ tissue with the methods
used. However, it is open to two objections, viz., the obser-
vations of Spuler, confirmed in a casual and not wholly satis-
factory way by Livini, that fibers actually lie within the cells,
and not always at the periphery, as Hansen’s and Mall’s theories
presuppose, but even in direct contact with the nucleus; and,
secondly, if the cells are in active amceboid motion, with loco-
motion as well, as is certainly the case in the fins of the embryo
Fundulus, then one can hardly conceive that the more or less
fixed syncytium described by Mall, whose observations can be
readily verified by any of the usual histological methods applied
to the tissue used, is sufficiently stationary to permit the retrac-
tion or shrinkage of endoplasm, thus leaving the endoplasmic
fibrils outside the bounds of the cellular protoplasm, for according
to-Mall’s theory true cells are lost, being replaced by nucleated
endoplasmic areas anastomosing with their neighbors to form a
continuous syncytial net.

I have many times observed the fins of Fundulus embryos
between the lengths of 4.5 mm. (at the time of hatching) and
25 mm., the pectoral and caudal fins being usually selected, and
in no single instance have I observed either a stellate or a spindle
cell which did not exhibit changes of form and locomotion, the
latter being sometimes very limited, at other times, as shown
in Fig. 2, quite extensive. Hence the ultimate theory of con-
nective tissue histogenesis, it would seem, must take into account
the relative activity of the mesodermic cells. So far as I ap-
preciate them, none of the theories thus far advanced fully
meet all of the observed conditions. Further studies of both
living and ‘‘fixed’’ tissue may further elucidate the true conditions
involved in the histogenic process.

The fins of living fish embryos offer for the development of
connective tissue a most desirable subject for study, for in the
subcutaneous tissue between the dermal fin-rays one finds a
definite connective tissue area, in which, with the exception of
the somewhat related chromatophores, there is no other structure

279 JEREMIAH S. FERGUSON.

to confuse the picture. This area is bordered on either side by
the dermal fin-ray, beneath which an efferent blood-vessel
descends, and alongside of which the return or afferent vessel
ascends the fin; it is covered only by the pavement cells of the
dermal epithelium, whose outlines are sharply defined, lying at
a more superficial focal plane than the connective tissue. The
chromatophores are easily recognized both by their color, black
to yellow, and by their peculiar branching form, so that even
those relatively or almost entirely devoid of color can be easily
differentiated from the smaller colorless connective tissue cells.
Moreover, the chromatophores are prone to lie in immediate
relation to the fin-rays and to the blood-vessels.

In conclusion I desire to emphasize the opinion that in the
study of histogenesis of connective tissue we must take into
consideration the conditions surrounding the living cells and
draw conclusions from “‘fixed’’ tissue only in the light of our
knowledge of the living.

To furnish conclusive results living cells must be observed
under normal conditions and not merely under those most un-
usual, or even pathological, conditions which surround the ob-
servation of growing tissues either in the mesentery studied by
Flemming and others, open to the criticism of the presence of
inflammatory changes, or in the tissue cultures by the more
recent method of Harrison, Carrel and Burrows, which is sur-
rounded by the necessity for the interpretation of results arising
under entirely new surroundings and conditions as yet but little
understood. Both in the mesentery and in tissue cultures
movements of the connective tissue cells and even locomotion
have been observed. I now add to these former results the record
of the observation of motion and locomotion in cells under wholly
normal conditions, in animals which underwent no operation
other than in some instances the administration of chloretone,
and which remained alive, active and normal for some time sub-
sequent to the period of observation. For example, the par-
ticular fish which furnished the subject for Fig. 1 was returned
to sea water and remained actively swimming for two days; he
could have been kept much longer.

For the opportunity of pursuing this study I am indebted
to the Marine Biological Laboratory at Woods Hole, Mass.

Reprinted from THe ANaTomicat REcorp, Vou. 5, No. 12
December, 1911

ON THE STROMA OF THE PROSTATE GLAND, WITH
SPECIAL REFERENCE TO ITS CONNECTIVE
TISSUE FIBERS

JEREMIAH S. FERGUSON
From Cornell University Medical College, New York City

FIVE FIGURES

The prostate gland consists essentially of secreting ducts and
alveoli embedded in a stroma of connective tissue containing much
smooth muscle. The smooth muscle is divisible into three por-
tions: (1) the inner, which surrounds the urethra and consists
of longitudinal and circular fibers; (2) the outer, which encapsu-
lates the gland and is likewise disposed in longitudinal and cir-
cular bundles; and (3) that intermediate muscle which lies between
the inner and outer divisions and whose fibers form interlacing
bundles having a general trend from without inward.

Between the bundles of the intermediate muscle the glandular
tissue is disposed in more or less slender conical lobules beginning
with a broad base formed by dilated acini in the outer portion of
the gland and tapering to an apex from which a duct leads inward
to finally open on the surface of the prostatic urethra in the region
of the colliculus seminalis.

The volume of smooth muscle in the framework or stroma is
considerable; roughly, it forms nearly one-half of the tissue which
is interposed between the glandular alveoli. Its fibers are coarse,
and are easily observed in all preparations. They are specially
distinct in sections which have been stained according to the iron-
haematoxylin method of Heidenhain.

The muscle is embedded in a delicate network of connective
tissue fibers which, unlike the usual arrangement, penetrate the
muscle bundles and form between their individual fibers a close
but delicate mesh.

THE ANATOMICAL RECORD, VOL. 5, NO. 12
DECEMBFR, 1911

544. JEREMIAH S. FERGUSON

more abundant in the narrow septa between the lobular acini.
In this location the meshes are frequently occupied by the ecapil-
lary vessels which occur with great frequency close beneath the
glandular epithelium (fig. 2).

It is usually stated that the acini of the prostate have no mem-
brana propria or basement membrane. KoOlliker® states that
beneath the prostatic epithelium a membrana propria is entirely
wanting. Oppel’ says that careful examination with the best
immersion systems failed to reveal any structure in the layer
beneath the epithelium. Sections of the prostate prepared with
the Bielschowsky method show beneath the glandular epithe-
lium of nearly all of the acini a very definite membranous layer,
consisting of closely interlaced 6-collaginous fibers, upon which
the epithelium directly rests (fig. 3, b.m.). On examining such
sections, even with very low magnification (e.g., x 60) one is
immediately impressed with the prominence of the membrane.
In haematoxylin-eosin sections this membrana propria, for such
we may call it, can scarcely be recognized; with iron-haematoxylin
it can scarcely be discerned and is not sharply differentiated;
with the Mallory connective tissue stain it ean be distinguished
but lacks prominence because of the absence of color differentia-
tion from the a-collaginous fibers.

The fibers of the 8-collaginous membrane of the mucosa are
continuous with the similar fibers which form the coarser net-
work of ‘white fibers’ between the walls of the glandular acini,
and whose meshes form the peculiar oval areas already described,
such areas as are characteristic of the 6-collaginous fibers wherever
found, and to which attention has already been drawn by Ciaccio®
in his description of the ‘perifollicular plexus’ about the nodules
of lymphoid organs. This peculiar conformation appears to be
due to a tendency of these fibers to pursue a spiral course. I
have observed this tendency not only in the prostate and in
lymphatic organs, but elsewhere, e.g., among the collaginous
fibers of the derma, and among the fibers of elastic tissue (liga-

® K6élliker Gewebelehre, 6te Aufl., Bd. 3, S. 470.

7 Oppel, Lehrb. d. vergleich. mik. Anat., Teil 4, S. 397.
§ Ciaccio, Anat. Anz., 1907, vol. 31, p. 594.

THE STROMA OF THE PROSTATE GLAND 545

mentum nuchae, arterial tunica intima, etc.); in this last location
their spiral character is most apparent (fig. 4).

The £-collaginous fibers closely invest the blood-vessels, the
capillaries as well as the adventitia of the arterioles and venules.
Oppel? has shown an excellent figure of the relation of these fibers
to the capillary vessels but without directing attention to their
‘peculiar character. The capillaries are enclosed within the oval
meshes formed by these peculiar fibers. The endothelium rests
directly upon the fibers.

The disposition of the 6-collaginous fibers within the muscular
tissue of the prostate is also peculiar and characteristic. The
larger bundles of smooth muscle are not always penetrated
throughout, but in the smaller bundles the 8-fibers form delicate,
rounded oval meshes, whose fibers are intricately interlaced, and
within the spaces of which are the individual muscle cells. Even
if a smooth muscle fiber lies isolated within the prostatic stroma
it will usually be found surrounded by the interlacing 8-fibers by
which it will be inclosed within an oval space (fig. 5). The
muscle fibers are not, however, over closely grasped by the
enveloping £-fibers, there being usually a narrow interval between
muscle and collaginous fiber.

SUMMARY

The method of Bielschowsky discloses within the prostatic
stroma, in addition to smooth muscle and elastic fibers, two
types of collaginous fibers, the one being of the wavy variety
usually designated as typical ‘white fibers’ though here they are
of rather unusual delicacy, and the second a type which we have
called the s-collaginous and which in successful Bielschowsky
preparations are characterized by an opaque black color reaction;
these last form a membrana propria supporting the glandular
epithelium, and an interlacing network throughout the intergland-
ular tissue. They closely invest the blood vessels and penetrate
between the individual fibers of the smooth muscle bundles.
That they are not elastic fibers is amply demonstrated by the

® Oppel, loc. cit.

~

546 JEREMIAH S. FERGUSON

fact that they do not react to the special stains for elastic tissue,
and by the additional fact that elastic fibers in known locations,
e.g., in the tunica intima of the arteries, or in the ligamentum
nuchae, remain colorless when treated in accordance with the Biel-
schowsky method.!°

The above study was partially conducted on sections which
had been prepared under my direction by Mr. I. Rosen, first
year student in the Cornell University Medical College, for whose
assistance I am greatly indebted.

‘ PLATE 1

EXPLANATION OF FIGURES

1 a-and 6-collaginous fibers from the stromaof the human prostate gland. The
6-fibers are coarse and black, the a-fibers much finer and of a golden brown color
in the original preparation. Bielschowsky stain. Drawn with Edinger projection
apparatus, < 900, reduced one-fourth in reproduction.

2 The epithelium and intervening stroma of adjacent prostatic alveoli. 8B,
6-collaginous fibers; bl. ves., blood-vessels; ep., secretory epithelium of the pros-
tate. Bielschowsky stain. Drawn with Edinger projection apparatus, X 270,
reduced one-fourth in réproduction.

3 Severalalveoli and the intervening stroma of the prostate gland. The base-
ment membrane is prominent, even with relatively low magnification. 06.m.,
basement membrane ep, epithelium of the prostatic alveob. Bielschowsky stain.
Drawn with Edinger projection apparatus, X 200, reduced one-third in repro-
duction.

4 Tsolated 6-collaginous fibers from the tunica intima of the human aorta, show-

ing their typically spiral course. Bielschowsky stain. Drawn with Edinger
projection apparatus, < 270, reduced one-fourth in reproduction.
5 The @-collaginous fibers of a muscle bundle of the prostaticstroma. At the
right of the fibers the smooth muscle fibers were transversely, at the left of the fig-
ure they were longitudinally cut. The muscle fibers are not represented; each
mesh at the right of the figure enclosed a single muscle fiber, and a single muscle
fiber occupied each interstice between adjacent 6-collaginous fibers at the left.
a-collaginous fibers, indicated by the finer and lighter lines are found between
muscle bundles and about the coarser §-fibers. L.S., portion longitudinally cut;
T’.S., portion transversely cut. Bielschowsky stain. Drawn with Edinger pre-
jection apparatus, X 270, reduced one-fourth in reproduction.

40 Ferguson, Am. Jour. Anat., vol. 12, no. 3, 1911.

STROMA OF THE PROSTATE GLAND PLATE 1
J. 5. FERGUSON

THE ANATOMICAL RECORD, VOL. 5, NO. 12

THE TEACHING OF VISCERAL ANATOMY,
OR ORGANOLOGY

JEREMIAH S. FERGUSON, M.S., M.D.
Assistant Professor of Histology, Cornell University Medical College

NEW YORK

Medicine as a science is yet in its infancy; its com-
posite development, its evolution, forms a_ steadily
increasing curve, but each of its component parts form
irregular curves with frequent rises, falls and stationary
levels, when historically considered. The close of the
last century marked the completion of a period of rapid
development in surgical science, during which it was,
perhaps, eminently “desirable that the medical student
should view anatomy with exaggerated reference to its
surgical importance.

The beginning of the present century offers promise of
an equally important development on the side of internal
medicine. The former necessity is now superseded by
the latter, and for the internist an accurate and exten-
sive familiarity with visceral anatomy is of fundamental
importance.

If there was a time when students could be allowed to
devote weeks to the careful dissection of an arm or a leg
and then for want of time pass cursorily over the
abdominal contents and perhaps fail entirely to open the
thorax, cranium or spinal canal, that time is past; the
demands of the internist in the present generation call
not only for careful visceral work in the dissecting-room
but for an amplification of that course by such methods
as shall result in accurate conceptions of living structure,
which in turn can result only from the familiarity
obtained through extensive first-hand acquaintance with
the tissues themselves. It is for the satisfaction of these
demands that courses modeled along the lines which I
am about to indicate must be offered to the medical
student of to-day.

2

The form, position and external relations of the
organs are best observed in the “fixed” material of the
dissecting-room, and if only recently prepared or care-
fully preserved tissue is used, the conception in the mind
of the student will be weli formed. But it is essential
that the tissue be perfectly preserved and that the dis-
section shall be so rapid and careful as to allow no
opportunity for excessive evaporation with coincident
changes of color, texture and size. Even at its best, pre-
served dissecting- room material will show considerable
changes in color and some hardening of texture.
Material which has lain for weeks on the table during
the dissection of the limbs and body wall, unless it has
received the best of care, is not entirely satisfactory for
the proper study of the viscera. ‘To obtain the best
results from the standpoint of organology a body freshly
prepared by injection should be brought directly from
the preparation room, its paries at once opened, as at
autopsy, and its viscera carefully surveyed, as rapidly as
is consistent with thoroughness, first for their position
and general relations and then for form and structure.
Such organs as are not badly altered by disease should
then be removed, their connections to paries and to
adjacent organs and especially their vascular connec-
tions being carefully noted ; then their surface markings
are to be carefully observed and their architecture thor-
oughly studied. Even at the loss of some material the
finer details of dissection involving the topographic
anatomy of the organ will be better “pursued on other
material.

But even at its best, dissecting-room material only
partially fulfils the requirements of an accurate concep-
tion of the living viscera. Accurate conceptions and
exact knowledge of any matter are obtained only by inti-
mate first-hand acquaintance with the material. Abund-
ance of opportunity for handling anatomic material
should be afforded the student; no source of supply
should be neglected. A course in organology is there-
fore incomplete unless it utilizes an abundance of fresh
tissue, which only the autopsy room can supply. Since
the nearest approach to the actual living organs obtain-
able for anatomic study is found in the human tissues at
autopsy, and in the fresh tissues of the lower animals,
both of these sources should be extensively utilized for
the purposes of organology.

3

’

The recently dead animal presents organs, to all
intents and purposes, in a living condition. In certain
cases their cells can be transplanted and caused to grow;
indeed, they may even be cultivated extraneously as has
been demonstrated by R. G. Harrison and others.

The fact that the lower mammalian organs differ
anatomically from the human is not an objection to their
utilization, for, indeed, the very differences may often be
used to broaden the conception of human structure. The
fact that the sheep’s kidney is lobulated, or that the
kidney of the horse or dog has but one pyramid does not
detract from the value of either in demonstrating the
structural relations and appearance of the living human
kidney, and if properly utilized they respectively eluci-
date a logical conception of the markings on the surface
and the contents of the sinus of the human organ.
Again, the objective study of the surface markings of the
adult human lung is difficult of interpretation without
reference to the child, the infant, and the lower animals.

But while lower animal tissues may be of great value
in the demonstration of certain structural features they
cannot be relied on as material for any great portion of
such a course as is under discussion. Human anatomy
must be primarily taught from human material. The
handling and study of “unaltered human material from
the autopsy room offers the most valuable instruction to
the anatomic student.

The utilization of this source of material cannot be
left till such time as the student may pursue his work
in the autopsy room under the instruction of the pathol-
ogist, for there he will be concerned, not with anatomic
details, but with pathologic lesions, and his facility in
pathologic study will largely depend on the accuracy and
fulness of his anatomic conceptions, and on his familiar-
ity with the structure and appearance of the normal
rather than diseased viscera.

The result of a pedagogic experience of some years
has convinced me of the fundamental importance of
anatomic instruction based on fresh human tissue. A
student of anatomy cannot see too much material of this
kind ; he should use it for most careful study.

Much normal tissue is removed at autopsy by the
pathologist; with proper cooperation it should find its
way, while still fresh, to the department of anatomy. A
course in organology based on such material, amplified

4

by the use of lower mammalian and other animal tis-
sues, by museum specimens carefully prepared to pre-
serve form, color, and to show the vital appearance, as
nearly as possible, of certain special features of anatomic
structure, and the whole broadened by coincident demon-
strations on dissecting-room material which has been
carefully and freshly prepared with organs fixed in situ,
offers to the student of anatomy a well-nigh ideal oppor-
tunity for developing an accurate conception of the
human body as it actually exists in a functionally active
condition.

That such a course is utopian is not true. The fact
that just such a course has been offered for the past
three sessions in the laboratories of the Cornell Univer-
sity Medical College demonstrates its feasibility. The
essentials are some time and energy on the part of the
corps of anatomic instruction, and a very little friendly
cooperation on the part of the pathologists. The time
demanded from the student is little, ‘because what is
consumed in actual work renders so rich a return in
improved knowledge of structure and of general, funda-
mental, anatomic characters that the work of the dis-
secting-room, and especially of the microscopic labora-
tory may thereby be very materially abbreviated. For
this reason such a course in organology is best taught in
connection with microscopic anatomy and the work so
correlated that the gross anatomy of the organ 1mme-
diately precedes its histology.

The consideration of the value of a course in organ-
ology on the above lines directs attention at once to cer-
tain phases of the relative value of the didactic and
laboratory types of instruction. The value of the former
type depends on the assumption that known facts are
more readily obtained by the novice through the recorded
observations of his predecessors, while “the laboratory
type presupposes that an accurate and utilizable knowl-
edge must result only from personal contact with the
object of study.

For this last type of work a progressively increasing
value is represented by the book, the model, and the
actual material. Given unlimited time and unlimited
material, a trained student has little need of other
sources of information. But for the great bulk of med-
ical students time in the laboratory is not unlimited;
hence the intensity of the laboratory work is greatly

5 :

enhanced if accompanied by ample opportunity for read-
ing and reflection in study hours. In the course which I
have hitherto offered, the work of the student is also
greatly facilitated by the use of a brief syllabus which
indicates the names of structures of anatomic interest,
without comment or description, arranged in the order
in which they are uncovered.

With such opportunity it is remarkable how rapidly
an accurate systematic conception of an organ may be
acquired. In the laboratories of the University of Chi-
cago it has been found that the study of the human brain
is not only simplified but accelerated by a preliminary
examination of the less complex nervous system of the
dogfish, and the course is therefore begun with the dis-
section of the selachian nervous system. A true concep-
tion of the general fundaments, the gross divisions of
structure as it were, renders the more intricate and com-
plicated system so much more readily mastered as to
result in an actual saving of time to the student as well
as in a more utilitarian knowledge of the subject.

Likewise, in my laboratory at the Cornell University
Medical College the survey of the fundaments of struc-
ture represented by gross organology as outlined above
rendered possible an actual shortening of the time
allotted to microscopic anatomy, and, far more import-
ant, I believe that the student better appreciates the full
structure of the organs so studied. It is a matter of
observation that he turns from his anatomy to his phys-
iology better prepared to apply his anatomic knowledge
to the deduction and explanation of function than was
possible under other conditions. When one has studied
in the gross the bundle of His and its ramifications, seen
it in the fresh tissue, as well as in the dissected specimen
and the microscopic section, and all have been properly
correlated by coincident study, he has a preparation for
the consideration of heart-block which cannot be obtained
by the isolated work of the dissecting-room or the his-
tologic laboratory. When those courses have been cor-
related by the study of the conduction system in fresh
tissue and the transference of bits of known location to
the stage of the microscope for verification of structure,
the student thus prepared is in full possession of the
anatomic mechanism, so far as it concerns the bundle in
question, on which the phenomenon of heart-block largely
depends,

6

A course in organology based on fresh material is,
therefore, closely related to the work of the microscopic
laboratory, and for him who pursues it the utmost of
value is only obtainable when the facilities of the
autopsy, the dissecting-room, and the microscopic labora-
tory have been coincidently placed at his disposal. With
the organization of broad-minded departments of anat-
omy with gross and microscopic work provided for under
the same head, an arrangement now generally adopted in
leading medical schools at home and abroad, such corre-
lation of work appears entirely feasible and eminently
desirable.

330 West Twenty-Eighth Street.

Reprinted from The Journal of the American Medical Association
May 27, 1911, Vol. LVI, pp. 1544-1546

Copyright, 1911
American Medical Association, 535 Dearborn Ave., Chicago

ERRATA

Page 132, first word, eighth paragraph, for Candy read Gandy.

Page 134, end of fourteenth line, for to read of.

Page 136, fifteenth line, for Doudena read Duodena.

Plate 1, explanation of figure 6, for ‘“‘This natural-size sketch represents

he doudenal mucosa,” ete., read ‘This sketch represents the duodenal
ucosa,”’ ete.

}
,

THE ANATOMICAL REcorRD, Vol. 5, No. 3, March, 1911.

Reprinted from THE ANATOMICAL ReEcorp. VOL. i, No. 3.
March, 1911.

DUODENAL DIVERTICULA IN MAN

WESLEY M. BALDWIN
Cornell University Medical College

TWENTY-SIX FIGURES

Generally speaking diverticula of the duodenum are rare in
comparison to the number of diverticula described in the litera-
ture as occurring elsewhere in the intestine. These have been
divided by most authors into two classes, congenital and acquired,
which are terms sufficiently explanatory in themselves. There
are also two sub-groups known as ‘true’ and ‘false,’ the basis of
this division being entirely anatomical. A ‘true’ acquired diver-
ticulum presents in its walls all coats of the normal intestine, 7.e.,
mucosa, submucosa, muscularis, and peritoneum (fig. 1). The
presence or absence of the last named constituent depends, how-
ever, upon the site of the diverticulum, namely, whether arising
from the mesenteric border of the gut and thus projecting between
the layers of the mesentery or whether situated along the convex
free surface of the intestine. On ‘the other hand, in the ‘false’
variety of acquired diverticula the muscularis is wanting, the walls,
then, being formed by mucosa and submucosa alone. Accordingly,
one might readily conceive of this form as being a hernia of the
mucosa with the submucosa through arent in the muscular layers
of the intestine, and this conception has been verified in some in-
stances by actual findings of diverticula in which the muscularis
ceased abruptly at the orifice of the sac (fig. 2).

This Cornell series of duodenal diverticula is remarkable for
two reasons: first, that so large a number as fifteen should be dis-
covered among a series of only 105 duodena, and, secondly,
because of the fact that the series includes the largest diverticulum
yet mentioned.

121

122 WESLEY M. BALDWIN

For purposes of convenience in the matter of description of
these specimens, I have divided them into the following classes
based upon their gross anatomical form. In fig. 3, a, represents a
funnel-shaped diverticulum, b, a tubular or cylindrical form, and c,
a spherical or globular diverticulum.

Further, with the idea of making this paper a complete presen-
tation of the subject of duodenal diverticula, I have collected the
descriptions of the specimens heretofore reported in the literature
and herewith present them in abstracted form.

The Cornell series

Specimen 1. (Figs. 4 and 5) This specimen (no. 353) was
removed from the body of a white male, age 65, weight 150
(estimated), who had died of senility. Six centimeters caudal to
the major duodenal papilla a wide-mouthed, funnel-shaped diver-
ticulum opened upon the cephalic surface of the third portion of
the duodenum. The sac extended cephalad a distance of 1.0
cm. lying dorsal to the head of the pancreas but did not penetrate
it. The fundus measured 2.0 cm. transversely by 0.7 cm. in the
dorso-ventral direction. The structure of the sac-wall was simi-
lar to that of the duodenum, all of the coats, however, being
reduced in thickness. There was no relation of the fundus
to any of the large vessels or to the ducts of the pancreas.

Specimen 2. (Figs.6and7) The history of this specimen was
not available (s795). In the descending portion of the duodenum
there was a large diverticulum with a circular orifice 1.5 cm. in
diameter. The fundus, globular in shape, extended medially a dis-
tance of 2.5 em., lay dorsal to the head of the pancreas and
possessed an approximate diameter of 2.0 cm. The bile and main
pancreatic ducts traversed the ventral wall of the sac, opening at
the ventro-caudal angle of its orifice. The minor papilla was not
related to the diverticulum. The mucosal lining presented several
folds, but the structure of the wall differed in no particular from
that of the duodenum excepting that all the layers were thinner
and the glands less numerous.

Specimen 3. (Fig. 8) White female, age 82, 5 feet, 2 inches
tall, weight 140 (estimated), died of dysentery (no. 287). In this

DUODENAL DIVERTICULA , 123

we

Fig.1 Inthisdiagram the intestine is represented in cross-section with a ‘true’
acquired diverticulum opening into it. The thin inner line represents mucosa,
the interrupted line, submucosa, while the heavy line represents the intestinal
muscularis. As is to be seen, all three layers are carried uninterruptedly over
the fundus of the sac.

Fig. 2 In this diagram a ‘false’ acquired diverticulum is figured opening into
the intestine. The muscularis, represented by the heavy outer line, ends abruptly
at the mouth of the diverticulum, the mucosa and submucosa, however, repre-
sented by the thin inner line and the broken line, respectively, constitute the
wall of the diverticulum,

124 WESLEY M. BALDWIN

subject a diverticulum of unusual size projected into the head of
the pancreas from the medial wall of the descending portion of the
duodenum. Its communication with the duodenum measured
4.0 em. in the long axis of the intestine by 2.5 cm. transversely,
being somewhat quadrilateral in shape. ‘The fundus, loosely
attached to the substance of the pancreas which encompassed it,
presented proportions approximately equal to those of the head of
the gland, 7.e., it measured 6.0 cm. cephalo-caudally, 3.0 em. dorso-
ventrally, and 4.0 cm. transversely. Within the sac a prominent
fold of mucosa 0.9 em. broad and 2.2 cm. long passed cephalo-
caudally across the dorsal wall. The bile duct, traversing this
fold at some distance from its free edge, terminated at the major
duodenal papilla 1.0 em. within the sac at the junction of its dorsal
and caudal walls. The main pancreatic duct passed dorsal to the
diverticulum to open at the major papilla with the bile duct.
Upon the duodenal mucosa, cephalad and ventral to the orifice
and about 1.0 em. from it, lay the minor duodenal papilla with the
opening of the accessory pancreatic duct, the latter traversing
the pancreatic substance of the head of the gland ventral to the
fundus of the diverticulum. The mucosa of the diverticulum was
smooth with but few villi, and devoid of intestinal valves and of
intestinal glands. A single thin layer of muscularis existed over
the fundus and became somewhat thickened at the orifice of the
sac. Numerous large blood vessels were to be seen distributed
along the parietes.

Specumen 4. (Figs. 9and 10) This specimen (no. 483) was re-
moved from the body of a white female, age 53, weight 98, diagno-
sis ‘heart disease.’ Lying directly cephalad and dorsal to the
major duodenal papilla in the second or descending portion of
the duodenum an orifice, measuring 0.7 cm. transversely and 0.5
cm. in the long axis of the intestine, communicated with a spherical
diverticulum. The fundus of this sac, with dimensions somewhat
more ample than those of the orifice, lay dorsal to the head of the
pancreas, to which it was adherent by loose connective tissue
fibrils, and extended medially a distance of 0.5 em. The sac bore
no immediate relation to the orifice of the accessory pancreatic
duct, but both the main pancreatic and bile ducts traversed its

DUODENAL DIVERTICULA , 125

ventro-caudal wall. No sphincter-like arrangement of the duo-
denal muscularis existed at the orifice of the sac. All of the ele-
ments of the duodenal wall were represented in the parietes of the
diverticulum.

Specimen 5. (Fig. 11) Male, age 55, white, weight 140 pounds
(estimated), height 5 feet 7 inches, diagnosis cerebral hemorrh-
age (no. 377). The medial wall of the descending portion of the
duodenum presented a quadrilateral orifice with sharp and well-
defined edges measuring 2.5 cm. in the long axis of the intestine
and 1.5 cm. transversely. This orifice opened into a large spherical
diverticulum which projected medially a distance of 2.5 cm. into
the substance of the head of the pancreas midway between its
dorsal and ventral surfaces: The fundus measured 3.0 em. cephalo-
caudally by 1.0 cm. dorso-ventrally. The bile duct in company
with the main pancreatic duct traversed the dorsal wall of the sac,
both opening at the major papilla which was situated 0.7 cm.
within the orifice upon the dorsal wall of the diverticulum. <A
prominent fold of mucosa bearing this papilla upon its crest ran
caudally across the dorsal wall. The minor papilla was located
upon the duodenal mucosa 1.2 cm. cephalad and ventral to the
diverticulum. Microscopically the fundus showed a thin muscu-
laris completely investing the sac. No sphincter was present.
The villi were more scattered over the fundus.

Specimen 6. (Figs. 12 and 13) The data in this case (s503x)
were not obtainable. At the common orifice of the bile and main
pancreatic ducts a globular diverticulum 1.5 cm. in depth and
1.2 cm. in the other dimensions, lying dorsal to the head of the
pancreas, opened into the duodenum through anirregularly quadri-
lateral orifice measuring 1.1 cm. in the long axis of the duodenum
and 0.4 cm. transversely. The ducts lay along the caudal wall
of the sac with their orifice at its caudal lip. With the exception
of the one-layered structure of the muscularis and the thinness of
the several coats in general, the constituents of the wall of the
sac did not differ from those of the duodenum. There was no
sphincter at the orifice.

Specimen 7. (Fig. 14) Female, age 61, white, height 5 feet 6
inches, weight 150 (estimated), death from sepsis (no. 341). The

126 WESLEY M. BALDWIN

figure represents a small, shallow, funnel-shaped diverticulum
upon the medial wall of the second portion of the duodenum pre-
senting in its depth the conjoined orifice of the bile and main
pancreatic ducts. The structure of the parietes of the depression
was precisely similar to that of the duodenal wall. This diverticu-
lum presented an orifice 0.4 cm. in diameter and a depth of but 0.2
em.

Specimen 8. (Figs. 15 and 16) Male, age 60, white, height
5 feet 8 inches, weight 160 (estimated), diagnosis endocarditis (no.
330). In this specimen a large diverticulum communicated
through a comparatively small orifice with the duodenum 4.5
cm. caudal to the major duodenal papilla. The body of the diver-
ticulum lay dorsal to the caudal portion of the head of the pan-
creas and measured 3.0 cm. in a cephalo-caudal direction, 2.0
cm. transversely, and 0.7 cm. in the dorso-ventral axis. The ori-
fice was irregularly oval, the long axis, parallel to that of the duo-
denum, being 1.0 cm. while the transverse axis measured 0.5 cm.
There existed at the orifice a sphincter-like ring of muscularis.
The walls of the sac were somewhat attenuated especially over
the fundus. The mucosa presented numerous folds representing
intestinal valves but did not differ in other respects from that of
the duodenum.

Specumen 9. (Figs. 17 and 18) Male, age 47, white, height
5 feet 6 inches, weight 140 (estimated), diagnosis ‘consumption’
(no. 363). At the summit of the duodeno-jejunal flexure a wide-
mouthed, funnel-shaped diverticulum extended cephalad a dis-
tance of 1.0 cm. and lay dorsal to the head of the pancreas.
Neither of the pancreatic ducts bore any relation to the diverti-
culum. The muscularis did not present a sphincter-like arrange-
ment at the orifice, nor did the structure of the walls differ in
any respect from that of the duodenal wall. No large vessels
were in association with the fundus of the sac.

Specimen 10. (Figs. 19 and 20) Male, age unknown, white,
height 5 feet 8 inches, weight 140 (estimated), diagnosis chronic
bronchitis (no. 305). This cylindrical diverticulum lay directly
cephalad to the major duodenal papilla, the common bile duct
passing along its ventro-caudal wall. The orifice, irregularly

DUODENAL DIVERTICULA ’ 17

round, measured approximately 0.4 cm. There was no sphinc-
ter. The diverticulum passed medially and cephalically a distance
of 1.0 cm. adjacent to the common bile duct and lying wholly
behind the head of the pancreas. The minor duodenal papilla
and the accessory pancreatic duct had no immediate relation to
the diverticulum. The walls presented the usual elements of the
intestinal wall, however, somewhat thinned.

Specimen 11. (Fig. 21) Male, age 59, white, height 5 feet,
weight 160 (estimated), death from ‘heart disease’ (no. 308).
This shallow diverticulum extended only 0.4 cm. from the cephalic
wall of the transverse portion of the duodenum towards but not
into the head of the pancreas. It lay 6.5 cm. caudal to the orifice
of the bile duct and its orifice measured 0.5 cm. in diameter.

Specimens 12 and 13. (Fig. 22) The data in this specimen
(s503) could not be obtained. There were two small, cylindrical
diverticula situated side by side on the same transverse plane
immediately cephalad to the minor papilla but with noimmediate
relation to the accessory pancreatic duct. Each measured 0.3 cm.
in width by 0.4 cm. in depth. The fundus of each was buried in
the tissue of the head of the pancreas. Their walls presented anor-
mal mucosa and submucosa; the circular muscularis was thick-
ened at the orifice to form a sphincter, the muscularis then be-
came thinner and formed a single stratum over the fundus.

Specumen 14. (Figs. 23 and 24) The data in the history of this
specimen (s804) could not be ascertained. Located upon the
cephalic surface of the transverse portion of the duodenum 6.0
cm. caudal to the major duodenal papilla a small tubular diverti-
culum 1.0 cm. deep lay dorsal to the head of the pancreas. This
diverticulum measured 0.5 cm. in transverse diameter and 0.3
em. dorso-ventrally. The orifice measured only 0.2 em. in the
long axis of the duodenum and but 0.1 cm. transversely. The mus-
cular stratum covering the fundus of the diverticulum was very
thin; in other respects, however, the wall did not differ from that
of the duodenum. Most of the muscle fibres covering the fundus
were derived from the circular layer of the intestine. The ducts
of the pancreas, of course, had no relation to the fundus of the
sac.

128 WESLEY M. BALDWIN

Specimen t5. (Figs. 25 and 26) Male, age 56, white, height
5 feet 7 in., weight 140 (estimated), death from dysentery (no.
365). Five centimeters caudal to the major duodenal papilla
upon the cephalic surface of the third portion of the duodenum a
large funnel-shaped diverticulum projected towards the dorsal
surface of the head of the pancreas. The side walls sloped gently
towards the fundus of the sac, whose maximum depth approxi-
mated 1.0em. At the orifice, where the dimensions were greatest,
the long diameter measured 2.5 cm. in the direction of the long
axis of the duodenum with the transverse dimension 2.0 cm. The
walls of the sac possessed all the coats of the duodenum, the mus-
cularis being represented, however, by only a thin stratum of muscle
fibres. The ducts of the pancreas bore no relation to the sac.

The essential facts of the Cornell series may be briefly stated as
follows. One duodenum presented two diverticula which were
located immediately cephalad to the minor duodenal papilla. Six
of the diverticula were in the pars inferior duodeni while
the other seven lay in immediate relation to the major duodenal
papilla. All of these diverticula were situated upon the left or
concave side of the duodenum extending towards the pancreas.
Four projected directly into the gland, eight lay behind, while
the other three were caudal to the head of the gland. None of
the diverticula presented any evidences of inflammatory condi-
tions. All of them belonged to the ‘true’ variety.

Following are the descriptions collected from the literature

While these abstracts have been made as brief as possible an effort
has been made to keep closely to the exact wording of the original in the
matter of the size, shape, etc., of the diverticula.

The first duodenal diverticulum mentioned in the literature was de-
scribed by Morgagni in 1761. This specimen, which was obtained from
the body of a man who had died of apoplexy, was situated two finger-
breadths caudal to the pylorus, the orifice being large enough to admit
a finger. The sac exhibited no traces of pathological changes.

Rahn (1796) found a duodenal diverticulum in the emaciated body
of a woman 22 years old who had died of chronic emesis. This sac-shaped
diverticulum was closely related to the pylorus and presented a mucosal
fold at its orifice not unlike that of the pylorus. The condition of gas-
troptosis was present in this cadaver.

DUODENAL DIVERTICULA ; 129

Fleischmann (’15) reported the following specimens:

Specimen 1. Male, 64, had used brandy freely, killed. The common
bile duct opened into a bladder-like diverticulum of the duodenum. Like-
wise the pancreatic duct opened intoa similar adjacent but smaller diver-
ticulum. The orifice of the larger diverticulum was devoid of intestinal
valves. The walls of these sacs were thin and lacking of muscularis
save at the orifice where a sphincter-like arrangement existed.

Specimen 2. Male, old age. The common bile duct opened into a
bladder-shaped diverticulum of the duodenum. Adjacent there were three
similar diverticula with lengths of a quarter, a half, and a full inch respec-
tively. Two of these were bladder-shaped; the third, and largest, was
constricted and pointed. The orifice of each presented a sphincter of
muscularis and a crescentric fold of mucosa. ‘The intestinal valves of
the neighborhood were deficient.

Specimen 3. Male, 28, drowned. The ductus pancreaticus and
the ductus choledochus opened separately each through a small duodenal
diverticulum. Lying near these diverticula there was a third of the size
of a pigeon’s egg and, towards the first portion of the duodenum, still
another smaller though similar diverticulum.

Albers (44) mentioned one diverticulum located in the horizontal
. portion of the duodenum. It was scarcely one inch long and presented
a contracted orifice with a marked fold of mucosa.

Barth (’51) reported in a female, aged 60, a duodenal diverticulum,
composed of all of the coats of the intestine, penetrating the head of the
pancreas and of a size sufficient to admit the little finger. This diverti-
culum seemed independent of the pancreatic ducts.

Habershon (’57) remarked having seen in Guy’s museum a duodenal
diverticulum near the orifice of the bile duct, but gave no further descrip-.
tion of the specimen.

Roth (’72) reported the following series of specimens:

Specimen 1. Male, workman, 50, dead from a blow on the head. In
the descending portion of the duodenum, 3 cm. cephalad to the orifice
of the common bile duct and 0.5 em. cephalad to the minor papilla, there
was a finger-like pouch 1.5 cm. long running caudal'y and medially into
the head of the pancreas. The fundus was covered ventrally by a thin
layer of pancreatic tissue with a thicker layer dorsally. The muscularis
existed only at the orifice in the form of a sphincter; elsewhere the walls
were constituted by mucosa and a thin layer of connective tissue.

Specimen 2. Male, 69, emaciated. At the level of the orifice of the
ductus choledochus, 7.e., in this specimen, the junction of the pars des-
cendens and pars transversus inferior duodeni, a cylindrical diverticulum
communicated with the duodenum through an orifice as large as a pea
and extended 1.5 cm. along the bile duct into the head of the pancreas.
Its walls were composed of thin mucosa and a thin layer of connective
tissue. A second diverticulum, similar in form, direction and extent of
long axis, structure, and in relations, existed 1.5 em. cephalad to it.
These diverticula were separated from each other by a thin layer of pan-
creatic tissue and by the common bile and pancreatic ducts. A promi-

130 WESLEY M. BALDWIN

nent fold of mucosa limited the constricted orifices. The muscle fibres
of the duodenum evidenced fatty degeneration.

Specimen 8. Male, 67, emaciated. Three centimeters cephalad to the
orifice of the common bile duct a diverticulum of the duodenum of the
size of a walnut penetrated the head of the pancreas. This rounded
diverticulum was composed of mucosa. Its orifice was of the size of a
lentil.

Specimen 4. Male, 58, emaciated. In the descending portion of the
duodenum there was a diverticulum of the size of a hazelnut.

Specimen 5. Female, 49, emaciated. In the descending portion of the
duodenum two thin-walled diverticula, one smaller and medial to the
orifice of the ductus choledochus, the other, more cephalad and as large as
a cherry, projected towards the head of the pancreas. The larger communi-
cated with the duodenum through an orifice sufficient to admit the little
finger.

Schiippel (’76) reported that he found a duodenal diverticulum pres-
ent in seven instances among 45 bodies studied. At Kiel, however,
among 200 bodies he found but one diverticulum.

Schirmer (’93) mentioned twospecimens. The first, 2.2 em. cephalad
to the orifice of the common bile duct, was 2.5 em. deep and 1.3 em.
broad; through the second, of the size of a pea, the accessory pancreatic
duct communicated with the duodenum.

Under the heading “‘ Large Pseudo-Diverticulum of the Duodenum,”’
Pilcher (94) described a pathological curiosity into which the duodenum
opened and from which the jejunum passed. The sac presented mucosa
in only one small strip. Elsewhere the parietes were devoid of epithelial
lining and exhibited inflammatory changes.

Good (’94) collected five specimens. Two of these received mere men-
tion, one being from the body of a female of twenty-seven who had died
of cholera nostras.

Specimen 1. Female, 77. Lateral and cephalad to the orifice of the
common bile duct a duodenal diverticulum of the size of a plum extended
into the head of the pancreas. The muscularis, deficient over the fundus
of the sac, existed at the constricted orifice in the form of a sphincter.

Specimen 2. Female, 54, died of carcinoma of the ovary with metas-
tatic involvement of the liver and peritoneum. One and a half centi-
meters cephalad and to the right of the orifice of the bile duct a thin-
walled cylindrical diverticulum 2.2 em. deep communicated with the
duodenum through an orifice 0.9 cm. in diameter. The bile duct passed
ventral and to the right of the sac while a layer of pancreatic tissue 0.5.
em. thick lay dorsal to it. The parietes contained no muscle fibres.

Specimen 3. Female, 51. <A spherical diverticulum 1.8 em. in dia-.
meter opened into the duodenum 3.5 em. cephalad and 1.4 em. medial to
the orifice of the bile duct and 10.0 cm. caudal to the pylorus. The fun-
dus projected medially a distance of 2.5 cm. into the substance of the
head of the pancreas, which separated it from the bile duct. No muscle

DUODENAL DIVERTICULA , 131

fibres were present in the wall of the sac nor did the mucosa present any
folds.

Seippel (’95) reported a cylindrical diverticulum of the duodenum at
the level of the major duodenal papilla, 5.0 em. in diameter and extending
about 3.0 em. dorsally and cephalically, the fundus being surrounded by
pancreatic tissue. Microscopically the wall of the sac revealed a mucosa,
somewhat thinned, a muscularis, and a muscularis mucosae. No other
muscle fibres were prolonged over the fundus of the sac, the muscularis
being represented there by a thin layer of connective tissue.

Hansemann (’96) reported a specimen of multiple diverticula in a man
of 85 who had died of pneumonia. He had been fat. The intestine con-
tained about 400 diverticula varying in size from a hemp-seed to a pigeon’s
egg. Several isolated examples were in the duodenum, but no descrip-
tion of these diverticula was given.

Helly (98) saw in a duodenum two diverticula, one located above
each papilla. The caudal of the two, corresponding to the major papilla
extended into the head of the pancreas.

Charpy (98) reported one specimen taken from the body of an aged
woman. The main pancreatic duct was small and terminated blindly at
a cul-de-sac of the duodenal wall. In another specimen a small diverti-
culum caudal to the orifice of the bile duct opened into the ampulla of
Vater but was not associated with either duct.

Letulle (’99) described two specimens. The first, of a depth of from
1.5 em. to 1.6 em., was composed of mucosa, with a few intestinal glands
and no duodenal glands, of muscularis mucosae and of connective tissue.
The muscularis of the duodenum ended abruptly at the orifice of the
diverticulum. In the other specimen five pouches were grouped about
the common orifice of the main pancreatic and common bile ducts, four
being cephalad and one caudal. These pouches extended medially to-
wards the head of the pancreas, but did not penetrate it.

Nattan-Larrier (’99) described a duodenum in which the common
bile duct emptied into a small cul-de-sac containing also the orifice of
the main pancreatic duct.

Marie (’99) says that he found in the body of a man of 45 a duodenum
which presented two diverticula. The larger extended about 5.0 cm.
medially, dorsal to the head of the pancreas and communicated with the
duodenum by a constricted orifice 2.0 em. in diameter. Both the bile and
the pancreatic ducts opened at the mouth of the sac. The other smaller
but similar diverticulum, 1.5 em. deep, lay more cephalad and opened into
the duodenum through a rounded orifice 1.0 em. in diameter. The muc-
ous lining of these diverticula was thin, smooth, devoid of villi, and of
duodenal glands, there remaining but little else than a layer of cylin-
drical epithelium. The muscularis of the duodenum stopped abruptly at
the orifice of the sac which it surrounded in the form of a ring.

Jach (99) made the following report:

Specimen 1. Female, 64. On either side of the major duodenal pap-
illa a diverticulum extended into the substance of the head of the pan-

132 WESLEY M. BALDWIN

creas with the bile duct lying between them. The larger or medial
of these two was as large as an egg, globular in shape and possessed
a somewhat constricted orifice sufficiently ample, however, to admit two
fingers. The pancreatic duct traversed its caudal wall. The smaller,
lateral diverticulum was as large asawalnut. Four centimeters caudal
to the major papilla two diverticula of the size of a cherry and with ori-
fices admitting the little finger, lay about 2.0 cm. apart, one being caudal
and medial to the other. Their dorsal surfaces were not covered with pan-
creatic substance.

Specimen 2. Immediately caudal to the pylorus acylindrical diverticu-
lum with a mouth large enough to admit the thumb extended 3.0 cm.
caudally, dorsally, and medially from the first portion of the duodenum.
Because of its cephalad position this diverticulum had no relation either
to the major duodenal papilla or to the head of the pancreas.

Specimen 3. Male, 58, carcinoma of the rectum. In the first portion
of the duodenum 2.0 em. from the pylorus there was an obliquely placed
cicatrix. Between this and the pylorus an orifice large enough to admit
one finger opened into a spherical diverticulum 1.0 cm. deep, the fundus
of which passed cephalad to the pylorus. Its dorsal surface was not
covered with pancreatic tissue.

Rolleston and Fenton (’00) described three specimens:

Specimen 1. Female, emaciated, hepatic cirrhosis. Two adjacent
duodenal pouches immediately cephalad to the orifices of the bile and
pancreatic ducts opened into the duodenum through separate but similar
orifices about 0.6 em. in diameter and extended medially into the head of
the pancreas. The bile duct traversed the fold of mucosa separating the
diverticula and opened into the duodenum apart from the main pancrea-
tic duct.

Specimen 2. This presented a pouch about 1.8 cm. cephalad to the ma-
jor papilla, 1.25 em. in diameter and buried to a depth of about 1.8
cm. in the head of the pancreas.

Specimen 3. This pouch was similar to specimen 2 and lay 1.25 cm.
cephalad to the major papilla, approximately 0.6 cm. deep and 0.6 cm. in
breadth. The walls of all of these sacs consisted of merely mucosa envel-
oped in loosely arranged connective tissue.

Gandy (’00) reported one specimen. Cause of death, strangulated
hernia. Nine centimetres caudal to the major papilla a saccular diverti-
culum opened into the dorso-cephalic surface of the third portion of the
duodenum through a circular orifice 1.0 cm. or 1.2 cm. in diameter. The
depth of the diverticulum was 1.5 em. the fundus being located at the
caudal border of the pancreas. The wall of the sac was composed of a
thin layer of mucosa devoid of folds and of a thin layer of connective
tissue.

LeRoy (?01) mentioned one specimen. Female, 64, emaciated. This
exhibited a cancer of the major duodenal papilla in association with a
diverticu.um. Dorsal to the papilla a spherical diverticulum 1.1 cm.
in diameter and 1.0 em. deep opened into the duodenum through a
circular orifice 0.9 em. in diameter limited by a mucosal fold. The wall

DUODENAL DIVERTICULA a 133

of the sac was composed of a much-thinned mucosa and a layer of con-
nective tissue. The muscularis spread out in a thin layer over the fundus
of the sac, which did not penetrate the pancreas.

Hodenpy1 (01) presented two cases of multiple spurious diverticula
of the intestine. In the first, the duodenum and the upper portion of the
jejunum were the seat of a number of thin-walled cysts varying in size
from that of a pea to that of an egg. Apparently no diverticula were
found in the duodenum of the second specimen.

Keith (’03) figured a case of ptosis of the pancreas from the London
Hospital Medical College Museum. Traction upon the common bile
duct had apparently been the factor in producing a diverticulum of the
duodenum at the major papilla. No further description was given.

Dorrance (’08) mentioned a diverticulum 3.5 em. caudal to the orifice
of the bile duct. The cavity measured 3.0 em. in depth by 5.0 cm.in
circumference. The walls were composed of thin mucosa and of a very
thin layer of circular muscle.

Bassett (’08) reported a specimen in a male, white, aged 78, who had
died of epithelioma of the lower lip. On the medial wallof the duodenum
10.0 em. caudal to the pylorus a diverticulum extended 2.0 em. cephalic-
ally and medially along the medial surface of the bile duct into the head
of the pancreas. The orifice, somewhat smaller than the fundus of the
sac, measured 2.0 cm. in the long axis of the gut and 1.0 cm. transversely.
The bile duct traversed the lateral lip, opening finally at the caudo-
lateral angle of the orifice. The mucous lining of the pouch was atrophic,
degenerated, and infiltrated in places with small round cells, the duo-
denal glands being absent. The muscularis ended abruptly at the orifice;
over the fundus the parietes consisted of mucosa alone.

Another specimen. Male, white, 78, miliary tuberculosis. This sac-
like diverticulum penetrated the head of the pancreas from the median
wall of the duodenum 10.0 cm. caudal to the pylorus, 3.0em. cephalad and
2.0 cm. medial to the opening of the bile duct. The rounded orifice
measured 1.5 cm. in the long axis of the duodenum by 1.0 cm. trans-
versely. Mucosa and submucosa, much folded and containing isolated
bits of pancreatic tissue, composed the wall of the sac. The muscularis
of the duodenum ended abruptly at the orifice of the diverticulum.

Jackson (’08) described a diverticulum of unusual proportions spring-
ing from the transverse portion of the duodenum in a male, aged 50,
who had died of pneumonia. The fundus of the sac lay dorsal to the head
of the pancreas and extended cephalically a distance of 3.5 cm., measur-
ing 3.0 em. transversely by 2.0 cm. ventro-dorsally, and communicated
with the cephalic wall of the duodenum through an orifice about 5.0 cm.
in diameter. There was no relation of the sac to the ducts of the pan-
creas. The wall of the diverticulum consisted of mucosa, a greatly hyper-
trophied muscularis mucosae, and a rudimentary muscularis.

Summing up, then, we may say that exclusive of the Hanse-
mann and Hodenpyl specimens, above mentioned, and of the

134 WESLEY M. BALDWIN

Cornell series, there are reports of only 67 specimens of duodenal
diverticula available in the literature. We believe, however,
that this does not represent the frequency with which these
anomalies occur in the dissecting room. Many elude observation,
others are noted but not reported, and again some are mentioned
in papers under irrelevant headings and consequently are with
difficulty collected from the literature. Schroeder, commenting
upon the Fleischmann series in 1854, remarked that there were in
the Fleischmann museum so large a number of duodenal diverticula
as to lead one to draw the conclusion that these anomalies were
found more frequently in the duodenum than in the remainder of
the intestine. Apparently, this completed series, however, was
never described. In this connection it is of interest to note that
the sac in several of these specimens lay ventral to the head to the
pancreas, a condition rarely seen by other observers. Schiippel,
as noted before, found seven instances among 45 specimens
examined, but in Kiel among 200 bodies examined with this end
in view, he found only one diverticulum. Fairland and Calder
both wrote of instances of duplication of the duodenum which
might be considered as diverticula very much elongated.

Much has been written concerning the etiology of these various
forms of diverticula. There has been little question over the
causative factors which have produced the Meckel’s diverticulum
of the adult, but the so-called ‘acquired’ diverticula have been
the occasion of much controversy. ‘False’ diverticula, which
belong to the class of ‘acquired’ diverticula, possess no muscula-
ture in their walls. These have been regarded by Seippel either as
a hernia of the mucosa betweén the muscle fibres of the intestinal
wall or as a localized bulging of the intestinal wall with a subse-
quent atrophy of that portion of the muscularis overlying the
fundus of the sac thus produced.

As causative factors in the instance of ‘true’ and of ‘false’
‘acquired’ diverticula, fatty degeneration of the muscularis,
atrophy of the pancreas, and ptosis of the duodenum have been
advanced by Roth, Jach, Keith, and others. Instances of the
production of diverticula through traction upon the duodenal

DUODENAL DIVERTICULA 7 135

wall have been cited by Hansemann, Birsch-Hirschfeld, Klebs,
and others. Increase in pressure from intestinal contents, liquid
or gaseous, has been cited and diverticula have been experimentally
produced in animal cadavers from these causes by Klebs, Edel,
Heschl, Good and Hanau,and Hansemann. These authors noted,
further, that in the instance of experimentally produced herniae
of the mucosa, theseat of hernia was almost invariably at the point
of entrance or emergence of the blood vessels, particularly the
veins. Since these results were more readily obtained in old maras-
mic subjects, the conditions found in the majority of diverticula
reported were thus apparently verified as, at least, contributing
factors. Chlumsky experimented upon the living animal bowel
and found, contrary to the results previously obtained with
dead intestines, that the rupture generally occurred at some point
opposite the mesentery. The traction effect of an accessory pan-
creas has received considerable attention and several observers
have reported instances where apparently this condition was the
sole etiological factor, 7.e., Zenker, Neumann, Weichselbaum,
Nauwerck, Hansemann, and others. Fleischmann says that the
production of diverticula at the major duodenal papilla is favored
by reason of the weakening of the duodenal wall at the point of
passage of the common bile and pancreatic ducts through the
muscularis.

In a paper upon the accessory pancreatic duct and the minor
duodenal papilla soon to appear, the author remarks that in 60
instances among 100 specimens of duodenum and pancreas exam-
ined, there existed at the major duodenal papilla a distinct hollow-
ing of the duodenal wall, the papilla with the orifices of the com-
mon bile and main pancreatic ducts in these cases being situated
in this depression. This condition is suggestive either of a per-
sistence of the original diverticulum from which the liver and the
ventral anlage of the pancreas developed, or illustrates the trac-
tion effect of the ducts upon the duodenal wall. Possibly, then,
these specimens of so-called ‘true’ ‘acquired’ diverticula are
in reality “‘congenital”’ to the extent that they represent a develop-

136 WESLEY M. BALDWIN

ment dating back to the time of hollowing of the duodenal buds in
the formation of the ducts of the liver and pancreas.

In a majority of the specimens reported in the literature, the
descriptions are so meagre that a complete analysis of the series
cannot be made. The following interesting facts, however, may be
presented. This tabulation includes the Cornell series.

Total number of duodenal diverticula:: .....- -.e see ee 8&3

In addition to this.number Hansemann reported one duodenum with several
diverticula and Hodenpy] two duodena, one with several diverticula. In the other,
the latter did not state clearly whether there were any duodenal diverticula.

Eliminating the Hansemann and the Hodenpyl specimens from the series and
considering that the 8 in the Schiippel series were single, 7.e., one diverticulum in
each duodenum, we may, with the restrictions mentioned above classify the
diverticula as follows:

Do%idena presenting:

One diverticulum=se es eee eee 48 Two diverticula: 1 soe 9
Three diverticulasse.. sels eee eo Houridiverticulasgss see eee aes
Rive, diverticula soseee eee eee 1

Total number of duodena (including Hansemann’s and Hodenpyl’s)... 63

Males. = eee 21 “Hemalese enc k eee 14> Notigiven..... = 2aeee

Available in 30 in- Youngest...... 22 (female) Oldest...... 85 (male)
stances
Two others were reported as ‘aged.’

Condition of cadaver

‘Emaciated’........ 1», HLiCHh Sakae eee 5 = *Medinmta aaa ae 5

Constituents of wall of diverticulum

With muscularis...20 Without muscularis...16 No report...........47

Position of fundus of diverticulum

Ventral to pancreas head....... Several of Fleischmann series (see above)
Penetrating pancreas~head..........-.......s.«<+. ++ oop 17
Dorsal to panereas head...........5).....2--+++ 02+.) ge 14

The remainder projected towards but not into the head of the pancreas.

DUODENAL DIVERTICULA 137

Location of diverticula

Reuse Gi AiOUCRUM |... . 2.2)... :4- Ae eee pete n--ahes-- 0
Concave (pancreas) side of duodenum ................-..02...2.5... 72
emit ages
Letulle.... 1
Specimens not described in full......................... { Hansemann. (?)
| Hodenpyl...(?)
bGoodie?...; 2
MO SEEME TU AERIS oo 2138 3s. oh Thy jak 2 eed ee ee Ee ee pet 2. 5
aa TRE TRIER RREAPLESENN SS 2 261063 5.0) oc" 2 1, A) Si ee Oe 9
With accessory duct opening into diverticulum....................... 1
eeSiCeer ar TATE: <8", etl ee ee ee ee ORI he F.- Aoeleas. 2 12
Pe MRESEIONS POISE 2 so ayia Sondre ees eee ee asa = oe A ie!
Bile duct opening into diverticulum: ..................006. cess eens 4
Main pancreatic duct opening into diverticulum...................... 3
(In one of these the duct was occluded).
Bile and pancreatic ducts together opening into diverticulum......... 4
Diverticulum in third portion of duodenum........................... 12

In conclusion I may say that it is a genuine pleasure for me here-
by to express my sincere appreciation of the helpful advice given
by Professors Gage and Kerr in the preparation of this paper and
also of the numerous and great courtesies shown by the depart-
ments of Anatomy and of Histology and Embryology.

138 WESLEY M. BALDWIN

BIBLIOGRAPHY
AvpEers 1844 Atlas Abt. 4. Taf. 21. Figs. 9 und 2. Erlauterungen zum Atlas
4.8. 262.
BartH 1851 Bull. de la Soc. Anat., vol 26, p. 90.

Bassett 1907-8 Duodenal diverticula; with especial reference to diverticula
associated with the pancreatic and biliary tracts. Tr. Chicago Path.
Soc., 7, p. 838.

Birca-HirscuFreip 1895 Lehrbuch der Path. Anat., 4 Aufl., S. 656.
CaupER 1733 Medical essays and observations, reprint, Edinburgh, 1, p. 205.

CuHarpy 1898 Variétés et Anomalies des Canaux Pancréatiques. Journ. de I’
Anat., p. 720.

Cutumsky 1899 Ueber versch. Methoden d. Darmvereinigung. Beitr. 2. klin.
Chir., Band 15,

DorrancE 1908 A diverticulum of the duodenum. Univ. of Penn. Med. Bull.
April.

EprL 1894 Ueber erworbene Darmdivertikel. Virchow’s Archiv. Bd. 138, §. 347.

FaIRLAND 1879 Congenital malformation of bowel; Amussat’s operation. Brit.
Med. Jour., 1, p. 851.

FLEISCHMANN 1815 Leichendffnungen, p. I, Erlangen.
Ganpy 1900 Bull. dela Soc. Anat. de Paris, Année 75, p. 691.

Goop 1894 Casuistische Beitrage zur Kenntniss der Divertikelbildungen u. s. w.
Inaug. Diss., Univ. Ziirich, p. 47.

HABERSHON 1857 Observations on the diseases of the alimentary canal. London,
p. 145.

Hanavu 1896 Bemerkungen zu der Mittheilung von Hansemann“ Ueber die Ent-
stehung falscher Darmdivertikel’’ in diesem Archiv, Bd. 144. Hft.
2, s.400, Virchow’s Archives, vol. 145.

HANSEMANN 1896 Ueber die Enstehung falscher Darmdivertikel. Archiv fiir
path. Anat., Bd. 144, Hft. 2, S. 400.

Hetty 1898 Beitrag zur Anatomie des Pankreas und seiner Ausfiithrungsgainge
Archiv fiir mik. Anat., p. 773.

Hescut 1880 Wien med. Wochenschr., no. 1, u. 2, spec. S. 5.

Hoprnpyt 1901 Two cases of multiple spurious diverticula of the intestine.
Proc. N. Y. Path. Soe. (1899-0), 182.

Jaca 1899 Ueber Duodenaldivertikel. Diss., Kiel. i

Jackson 1908 An unusual duodenal diverticulum. Jour. of Anat. and Phys-
iol., vol. 42.

DUODENAL DIVERTICULA 139

Keito 1903 On the Nature and Anatomy of Enteroptosis (Glénard’s disease).
Lancet, London, vol. 1, p. 640.

Kuess 1869 Handbuch d. path. Anat., Bd.1. 8. 271, Berlin.
1899 Die allgemeine Pathologie, theil 2, S. 100, Jena.

LeRoy 1901 Divertikel prévatérien congénital et cancer de l’ampoule de Vater
déterminant une obstruction biliare. Jour. des Sciences Med. d. Lille,
2, p. 598.

LETULLE 1899 Malformations duodenalés; diverticules péri-vatériens. La Presse
Medical, p. 13.

MariE 1899 Bull. et Mem. Soc. Anat. de Paris, p. 982.
Moreacni 176] De Causs. et sed. morb. Epist. 34. par., 17.

NattTan-LARRIER 1899 Malformations multiple, retrecissement du duodénum,
dilatation de l’oesophage, communication interventriculaire. Bull. et
Mem. Soc. Anat., Paris, p. 981.

Nauwerck 1893 Ein Nebenpankreas. Ziegler’s Beitrage, Bd. 12, S. 28.
NeumMann 1870 Archiv d. Heilkunde, Bd. 11, S. 200.

PitcHER 1894 Large pseudo-diverticulum of the duodenum. Annals of Surgery,
vol. 20.

Raun 1796 Scirrhosi pancreat. diagnos. obs. 14. Géttingae.

ROLLESTON AND FENTON 1900-01 Two anomalous forms of duodenal pouches.
Jour. Anat. and Physiol., vol. 35, p. 110.

Rota 1872 Ueber Divertikelbildung am Duodenum. Archiv. fiir Path. Anat., 41,
p. 197.

ScoirMER 1893 Beitrag zur Geschichte und Anatomie der Pankreas. Inaug. Diss.,
Basel, p. 58.

ScHROEDER 1854 Ueber Divertikelbildung am Darmcanale. Inaug. Dis., Erlang.
Scutrret 1876 Ziemmsen Handbuch der spec. Path. und Ther.

SrirpPeL 1895 Ueber erworbene Darmdivertikel. Inaug. Diss., Univ. Ziirich, p. 21.
WEICHSELBAUM 1883 Bericht d. K. K. Krankenanstadt, Rudolph-Stift, Wien. 4.

ZENKER 1861 Nebenpankreas’ in der Darmwand. Virchow’s Archives, Bd. 21,
S. 369.

THE ANATOMICAL RECORD, VOL. 5, NO. 3

PLATE 1
EXPLANATION OF FIGURES

4 This sketch, made from the actual specimen, represents the duodenal mucosa
with the mouth of the diverticulum. The duodenum was opened along that bor-
der opposite to the orifice of the diverticulum.

5 In this diagram the diverticulum shown in fig. 4 is represented as seen from
the dorsum. The fundus is represented projecting cephalically from the third
portion of the duodenum and lying dorsal to the head of the pancreas.

6 This netureksize sketch represents the doudenal mucosa with the orifice of
the diverticulum. The duodenum was opened opposite to the site of the diver-
ticulum. Note the close relation of the orifice to the major duodenal papilla.

7 In this diagram the dorsal surface of the head of the pancreas is shown en-
circled by the duodenum. The fundus of the sac represented in fig. 6 is seen pro-
jecting in close proximity to the bile duct and dorsal to the head of the pancreas.

8 In this sketch the mucosa lining the interior of the diverticulum can be seen.
Just within the orifice and running cephalo-caudally across the dorsal wall of the
sac a prominent fold of mucosa is seen presenting on its summit caudally the
major papilla. This diverticulum is the largest yet described.

9 Inthis sketch the close relation of the orifice of the diverticulum to the major
duodenal papilla is represented.
10 This diagram presents the relation of the fundus of the diverticulum, rep-

resented in fig. 9, to the dorsal surface of the head of the pancreas and especially
to the bile and pancreatic ducts.

REFERENCE NUMBERS

Major duodenal papilla.
Ductus choledochus.
Mucosal fold.

Ductus pancreaticus.

Orifice of diverticulum.
Duodenum (dorsal aspect).

Head of pancreas (dorsal aspect).
Diverticulum.

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DUODENAL DIVERTICULA IN MAN
WESLEY M. BALDWIN

THE ANATOMICAL RECORD, VOL. 5, No. 3

PLATE 2
EXPLANATION OF FIGURES

11 This sketch resembles in general appearance fig. 8. The fold of mucosa
traversing the dorsal wall,of the sac bearing the major duodenal papilla is well
shown. These structures lie just within the orifice of the diverticulum.

12 This sketch shows the close relation of the orifice of the diverticulum to the
major duodenal papilla.

13 This diagram shows the relation of the diverticulum, represented in fig. 12.
to the dorsal surface of the head of the pancreas and to the bile and main pancrea-
tic ducts.

14 In this sketch the common orifice of the diverticulum, the bile, and the main
pancreatic duct is shown upon the mucosa of the second portion of the duodenum.

15 This sketch shows the orifice of a diverticulum situated in the third por-
tion of the duodenum.

16 In this diagram the fundus of the diverticulum, represented in fig. 15, is
shown in relation to the dorsal surface of the head of the pancreas. There is, of
course, no relation to the bile or pancreatic ducts.

REFERENCE NUMBERS

1 Orifice of diverticulum. 4 Diverticulum.
2 Duodenum (dorsal aspect). 5 Major duodenal papilla.
3 Head of pancreas (dorsal aspect). 6 Ductus choledochus.

8 Ductus pancreaticus.

DUODENAL DIVERTICULA IN MAN
WESLEY M. BALDWIN

15

THE ANATOMICAL RECORD, VOL. 5, NO. 3

PLATE 3
EXPLANATION OF FIGURES

17 This sketch represents the duodenal mucosa exposed to show the orifice of
the diverticulum.

18 In this diagram the head of the pancreas encircled by the duodenum (fig. 17)
is represented as viewed from the dorsum. The fundus of the diverticulum
located at the duodenal-jejunal flexure is shown in relation to the pancreas.

19 In this sketch the orifice of the diverticulum lies immediately cephalic to
the major duodenal papilla.

20 This diagram represents the relation of the fundus of the diverticulum,
shown in fig. 19, to the bile and main pancreatic ducts and to the dorsal surface
of the head of the pancreas.

21 In this sketch the orifice of the diverticulum is shown upon the mucosa of
the third portion of the duodenum.

REFERENCE NUMBERS

1 Orifice of diverticulum. 4 Diverticulum.
2 Duodenum (dorsal aspect). 5 Major duodenal papilla.
3 Head of pancreas (dorsal aspect). 6 Ductus choledochus.

8 Ductus pancreaticus.

DUODENAL DIVERTICULA IN MAN
WESLEY M. BALDWIN

’ PLATE 3

THE ANATOMICAL RECORD, VOL. 5. No. 3

= PLATE 4
EXPLANATION OF FIGURES

22 In this sketch the relation of the small tubular diverticula to the minor
duodenal papilla is seen.

92

23 This sketch represents the mucosa of the third portion of the duodenum with
the orifice of the diverticulum.

24 In this diagram the fundus of the diverticulum, represented in fig. 23, is
shown as seen from the dorsum. The fundus extends cephalically lying dorsal
to the head of the pancreas but it has no immediate relation either to the ducts
of the pancreas or to the bile duct.

25 In this sketch a large diverticulum is represented opening into the third
portion of the duodenum.

26 This diagram represents the fundus of the diverticulum, shown in fig. 25,
as seen from the dorsum. It lies dorsal to the head of the pancreas but has no
relation to the ducts of the gland or to the bile duct.

REFERENCE NUMBERS
1 Orifice of diverticulum. 3 Head of pancreas (dorsal aspect).

2 Duodenum (dorsal aspect). 4 Diverticulum.
5 Minor duodenal papilla.

DUODENAL DIVERTICULA IN MAN ’ PLATE 4
WESLEY M. BALDWIN ;

THE ANATOMICAL RECORD, VOL. do, No. $

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Reprinted from THE JOURNAL OF ANATOMICAL RECORD, VOL. 5, No. 5.

THE PANCREATIC DUCTS IN MAN, TOGETHER
WITH A STUDY OF THE MICROSCOPICAL STRUC-
TURE OF THE MINOR DUODENAL PAPILLA

W. M. BALDWIN
Cornell University Medical College

TWELVE FIGURES

As ordinarily described in the text-books, there exist in the
substance of the pancreas, two ducts; one, the larger and more
constant, called the ‘pancreatic duct’ or ‘duct of Wirsung,’ and
the other, smaller and comparatively inconstant, the ‘accessory
pancreatic duct’ or ‘duct of Santorini.’ The main duct, beginning
in the tail of the pancreas, courses from left to right through the
body and neck to the head of the gland where it bends caudally,
after receiving the accessory duct, and, traversing the head of the
gland, perforates the duodenal wall to empty into the duodenum
in company with the common bile duct; occasionally, however,
apart from it. The accessory duct, on the other hand, is confined
to the cephalo-ventral segment of the head which it traverses
from its point of junction with the main duct to the minor duo-
denal papilla where it either empties into the duodenum or ter-
minates blindly. This minor papilla in the duodenal mucosa
bears a cephalo-ventral relation to the major papilla containing
the ampulla of Vater and lies about 1.8 em. from it. The one
noticeable feature of the anatomical descriptions of these parts is
the discordance of opinion concerning the terminal relations of the
accessory duct.

In the year 1641, Moritz Hoffmann discovered the duct of the pan-
creas while working on a rooster and showed his findings to Wirsung,
who the following year dissected the duct in the pancreas of a human
body. In a letter to Jean Riolan, Jr., Professor of Anatomy in Paris,
Wirsung gave to the world the first account of his important discovery.

THE ANATOMICAL RECORD, VOL. 5, No. 5
MAY, 1911
197

198 W. M. BALDWIN

Wirsung had the duct reproduced on a copper plate from which but
few copies were struck off. According to Choulant only two copies are
known to be preserved. Schirmer (1893) saw one in the University of
Strassburg and had a photolithographic reproduction of it made.

To Jo. Dominici Santorini belongs the credit for the first description
of the accessory pancreatic duct and for the first representation approxi-
mating accuracy of the arrangement of the ducts in the adult human
pancreas. He called attention to the existence of two papille in the
duodenal mucosa and figured them in table 12 of his published work.
In this connection, also, mention should justly be made of the name of
Regner de Graaf, who previously had reported that, contrary to what had
been the prevailing opinion, the pancreas might present two or even three
ducts.

A complete list of the workers upon this particular problem in con-
nection with the pancreas is lengthy; it includes such names as Vesling,
who reported two ducts, apparently in lower animals, Thomas Bartho-
linus, Bernard Swalwe, G. Blasius, Johannes von Muralt, and Chris-
tianus Ludovicus Welschius. Now that the identity of the ducts was
established, investigators began to report anomalous conditions of these
passages; as Albrecht von Haller, Tiedemann, Mayer, and M. Bécourt.
J. F. Meckel’s was a significant statement in explanation of the causative
factors involved in the production of the numerous anomalous condi-
tions observed, 7. e., that atrophy of the duodenal end of the accessory duct
was the developmental rule. A further list of workers at this period in-
cludes such names as Huschke, Jean Cruveilhier, and Sappey. Since the
time of Claude Bernard, who in 1846 revived interest in the accessory
duct, which had apparently been neglected, much attention has been
given to the relation of the accessory duct to the main duct and to the
duodenum. An incomplete list of the investigators thus engaged with
the number of specimens studied is as follows: Bécourt, 32; Verneuil,
about 20; Henle, Sappey, 17; Hamburger, ‘mehr als 50’; Schirmer,
105; Schieffer, 10; Helly, 50; Charpy, 30; Letulle, 21; Opie, 100.

A study of the different methods employed by the investigators in
their efforts to ascertain the condition of patency or occlusion of one or
both ends of the accessory duct is of two-fold interest, because it illustrates
the ingenuity of the workers, and, secondly, gives a probable explanation
of the inharmonious results of their work.

For example, Claude Bernard used injections of metallic mercury
which he forced into the main duct; Sappey, likewise working with mer-
cury, ligated the ampulla and injected through the common bile. duct.
Schirmer, however, availing himself of Henle’s objection to the use of
mercury as an injection fluid for reason of its liability to burst through
what might be a natural barrier at the blind duodenal end of the accessory
duct, had recourse to the ingenious method of blowing air at a low pres-
sure through the duodenal orifice of the main duct while the whole gland
was submerged in water. Charpy used the injection method. His
fluids were alcohol and some coagulable fluids, followed in some instances
by the air injection method. Taking his cue from Charpy’s comment

PANCREATIC DUCTS AND MINOR DUODENAL PAPILLA 199

upon Schirmer’s method in which the former said that it was well to dis-
lodge by friction the mucous which might obstruct the ducts, Helly
went one step farther towards accuracy by subjecting the minor papilla
to a microscopical examination, after he had injected the ducts with a
gelatin mass. His findings substantiated the previously advanced objec-
tions. Several times the injection mass broke down a natural barrier
thus giving rise to the erroneous conclusion, had the microscopical exami-
nation not followed, that the channel in life had been patent. On the
other hand, Helly found that several times a small accumulation of
mucous was sufficient to completely block the accessory duct.

This present work comprises a study of one hundred specimens
of adult human pancreas removed, with the exception of four
derived from autopsies, from the bodies used in the regular dis-
secting courses in anatomy in the Cornell University Medical
College at Ithaca, New York. The bodies had been embalmed
with a mixture of equal parts of carbolic acid, glycerin, and 95
per cent ethyl alcohol. The ages of the individuals ranged from
21 to 95 years. There were 57 males and 21 females plus a series
of twenty-two specimens from which the identification tags had
been lost and consequently all data. Death in no instance had
been caused by pathological processes localized either in the pan-
creas or in the duodenum.

The method followed in the examination of the specimens was
as follows: the ductus pancreaticus was located by gross dissec-
tion, with the aid of a lens magnifying two diameters, in the neck
of the gland where the pancreatic tissue overlies the superior
mesenteric vessels. At this level the duct approaches the dorsal
surface of the gland and is readily found usually about midway
between the cephalic and caudal borders at a depth of 2 or 3 mm.
in the gland substance. Once located, the duct was easily traced
both towards the tail of the gland and towards the duodenum.
In both of these regions it was found to lie nearer the dorsal than
the ventral surface of the gland.

The junction with the accessory duct was most quickly reached by
working along the ventral surface of the main duct beginning at
the neck and proceeding towards the duodenum. In those anoma-
lous instances where this duct could not be located by this method,
the duodenum was opened along its right border and the position

200 W. M. BALDWIN

of the minor papilla ascertained. Then, using this as a guide,
the accessory duct was sought for in the glandular tissue cephalad
to the level of the papilla. In those instances where no junction
of the accessory duct and the main duct could be readily ascer-
tained upon gross dissection, a ligature was passed around the
duodenal end of the accessory duct at the point of perforation of
the duodenal wall, and the main duct injected with a stain, either
aqueous eosin or methylene blue. Regurgitation of the fluid into
the accessory duct evidenced the presence of a communication
between the two ducts. However, in no instance was the acces-
sory duct injected with air or any fluid as a means of ascertaining
the condition of patency of its duodenal termination.

The relation of the ducts to each other being thus established,
the minor papilla entire, including the adjacent duodenal wall
and a small portion of pancreatic tissue, was imbedded in paraffin,
sectioned in series, and stained with the hematoxylin and eosin
method. In four instances the accessory duct was of such a large
calibre as to be readily followed through the papilla by gross
dissection. In these few specimens the papilla was not sectioned
and studied microscopically.

The relation of the main pancreatic duct to the termination of
the bile duct was studied by gross dissection, while by slitting
open both ducts their part in the formation of the major duodenal
papilla was ascertained. The major papilla, however, was not
sectioned or studied under the microscope.

In an investigation of this nature covering so much ground and
productive of so many data it has seemed wise to present the facts
of the problem in the topical order herewith listed.

1. The duodenal mucosa; its papillae and intestinal valves.

2. The main pancreatic duct; course, tributaries, and drainage.

3. The duodenal termination of the pancreatic duct in the
major papilla and its relation to the bile duct.

4. The accessory pancreatic duct; course, tributaries, and
drainage.

5. The minor papilla; relation to the accessory duct and micro-
scopical structure.

PANCREATIC DUCTS AND MINOR DUODENAL PAPILLA 201

6. The relation of the main pancreatic duct to the common
bile duct at the duodenal wall.
7. The bile duct and the major papilla.

1. The duodenal mucosa; its papille and intestinal valves

Attention was given to a study of the arrangement of the intes-
tinal valves. These were exposed by laying open the duodenum
along its convex border. The minor and major papille were
present in every instance of this series of one hundred specimens.
Locating the major papilla presented but little difficulty. On the
other hand, it was occasionally only as the result of the most
careful search that the minor papilla could be identified. It
lay cephalad and on a plane ventral to the major papilla in ninety
of the specimens. In eight instances the two papillae were on the
same vertical plane, the minor papilla being cephalad. Finally, in
the two remaining specimens the minor papilla lay upon the same
transverse plane with the other papilla but ventral to it. The
fact is worthy of special mention that in no instance did the minor
papilla occupy a position either caudal to the major papilla or
dorsal to it. Separating these papilla, the average distance,
measured from center to center, was 2.0 em. The shortest dis-
tance observed was 0.9 cm., and the longest 3.5 em., and the mean
distance 2.1 cm.

One specimen presented three papill ; the minor papilla oceupy-
ing the usual position relative to the major papilla and 2.3 em.
from it. The accessory papilla lay 1.0 em. directly cephalad to the
minor papilla. This third papilla had no pancreatic duct opening
through it.

Notwithstanding the apparently hap-hazard and chance disposi-
tion of the smaller and incomplete mucosal folds in the vicinity
of the papillz, there could be identified in these specimens a
marked conformity of the larger intestinal folds or valves to a
fixed and entirely characteristic arrangement. In order that a
more intelligible description might be made of these valves, I have
divided them into two classes, 7. e., ‘primary’ and ‘secondary.’

i)
=)
Ls)

W. M. BALDWIN

Cephalic end

sansa

Fig. 1 (natural size) represents the typical distribution of the ‘primary’ and
‘secondary’ folds of duodenal mucosa in the region of the two papille in the de-
scending portion of the duodenum. M.P.Minor papilla. P. Depression containing
the major papilla with the orifices of the bile and the pancreatic duct. C. Plica
longitudinalis duodeni. A,B,D. ‘Primary’ folds. S,S,S, ‘Secondary’ folds.

The basis of this classification is dependent entirely upon the size
and constancy of the folds (fig. 1).

The minor papilla (M.P.) occupies a position upon a promi-
nent ‘primary’ transverse fold or valve (A) and often at its bifurea-
tion as represented in the drawing. It lies not on the ridge or
crest of the valve but within the angle of bifurcation on the side
of the fold. About 0.5 em. caudal to this, a second, also ‘primary,’
fold (B) traverses the duodenal wall. Beginning at this second
valve a prominent ‘primary’ longitudinal fold (C) proceeds caud-
ally at a right angle and in the direction of the long axis of the duo-
denum. This is the ‘plica longitudinalis duodeni” of the text-
books. Upon its summit and close to its cephalic extremity,
in fact, overlapped by the fold indicated at (B), lies the major
papilla presenting the orifices of the bile and pancreatic ducts.
This plica passes caudally uninterruptedly across two or three
‘primary’ and ‘secondary’ (S) folds to terminate in another

PANCREATIC DUCTS AND MINOR DUODENAL PAPILLA 203

‘primary’ transverse fold (D) located at the junction of the de-
scending and transverse portions of the duodenum. Scattered
among these ‘primary’ plice are many small ‘secondary’ folds (S),
which haveno definite or constant arrangement, which branch often,
and which join or fuse with the larger folds at varying angles.
In addition to these features the observation was made upon
thirty of the specimens that if the valves were disregarded, ironed
out, so to speak, and merely the general contour of the duodenal
wall considered, at the level of the major papilla a distinct bulging
or hollowing of the wall towards the head of the pancreas was
demonstrable. This feature was superadded to the constant
dorso-ventral curvature of the duodenal wall and existed as a dis-
tinct entity. Furthermore, in sixty of the one hundred specimens,
the major papilla occupied a small, localized, but, nevertheless, dis-
tinct pitting of the duodenal wall (P). This pitting was produced
by simply a localized exaggeration of the general hollowing of the
medial wall of the duodenum mentioned above. To the mind
of the author, however, this hollowing was suggestive of a pos-
sible persistence of the original diverticulum from which in the
embryo both the liver and the ventral pancreas developed.

2. The main pancreatic duct; course, tributaries, and drainage

In order clearly to understand the arrangement of the
ducts and their accessory features in the adult, our consideration
must be turned to the embryology of the pancreas, which has
engaged the attention of many workers, including the names of
His, Phisalix, Zimmermann, Felix, Hamburger, Janosik, Jankelo-
witz, Swaen, Helly, Vélker, Kollmann, Ingalls, and Thyng.
According to the results of these investigators the human pancreas
develops from the duodenum at the level of the hepatic diverticu-
lum from two buds or anlages, one ventral, and the other dorsal,
the former being in association with the hepatic diverticulum.

Diverse views are entertained at present concerning the duplicity
of the ventral anlage, some maintaining that the bud is single from
the first while others hold that at the beginning it consists of two
lateral halves which subsequently fuse. The author has recently

204 WwW. M. BALDWIN

published a paper descriptive of an unusual form of adult pancreas
which possibly exemplifies a persistence of the earlier embryo-
logical condition of the primitive anlages.

Concerning the dorsal anlage we may say that contrary to what
had been previously demonstrated by Hamburger, Felix, and Jano-
sik, Thyng’s studies seem to prove that ‘‘the dorsal pancreas
arises from the intestine distinctly anterior to the hepatic diverti-
culum.”” From the dorsal bud the cephalic portion of the head and
all of the neck, body, and tail of the pancreas develop, the ductus
pancreatis dorsalis draining these portions. The ventral pancreas

Head Neck Body Tail
Portion
Accessory Developing
Pancreatic from
one Dorsal

Bile Duct Anlage
ti : :
Laie ; Portion Developing from

Ventral Anlage

enclosing its duct, the ductus pancreatis ventralis, forms ulti-
mately the caudal portion of the head of the gland (fig. 2).

The accompanying figure (2) represents diagrammatically
the parts of the gland derived from the two anlages. The clear
portion traversed by the heavy unbroken line is developed from
the dorsal anlage, while the shaded portion is derived from the ven-
tral anlage. The terms suggested by Thyng ‘ductus pancreatis
ventralis’ and ‘ductus pancreatis dorsalis’ are particularly appli-
cable from the standpoint of their embryological relations.

As development progresses, however, the ducts unite as is
shown in the sketch, the duct of the dorsal anlage then undergoing
a certain degree of atrophy at its duodenal end to thus produce
the adult arrangement (see also fig. 3).

205

D MINOR DUODENAL PAPILLA

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By keeping these facts of embryology in mind the anatomical
findings of this investigation have a much clearer interpretation.
The main duct was observed to begin in the tail of the gland through
the convergence of a number of small duct radicles. It could not
be demonstrated that these conformed to any particular arrange-
ment. Pursuing a more or less tortuous course, the duct passed
thence from left to right, traversing the glandular substance of
the body of the pancreas and approximating the dorsal rather than
the ventral surface of the gland. Furthermore, the duct lay nearer
the cephalic than the caudal border of the body.

Upon arriving at the head of the gland, the main duct inclined
somewhat abruptly caudally and dorsally with the convexity
towards the right and approached the dorsal surface of the head of
the gland. Reaching the level of the major duodenal papilla, the
duct now ran almost horizontally to the right to join with the
caudal aspect of the bile duct and empty with it into the major
papilla (fig. 3). |

The tributaries of the main duct in the body of the gland were
observed to join that duct almost invariably at right angles and
also to alternate with tributaries of the opposite side in the level
at which they joined the main duct. These same features in
turn characterized somewhat less noticeably, however, the radicles
of these tributaries of the main duct. Only in the head of the gland
was the conformity to these rules departed from. Here the tribu-
taries were, occasionally, of some irregularity, first, in the angle
of junction, and secondly, in the arrangement of the radicles.

Here also there existed one large unpaired trunk quite variable
in appearance but well represented in fig. 3. This is the chief
channel of drainage of the small lobe of the head (lobe of Winslow)
which lies dorsal to the superior mesenteric vessels. Winslow in
1732, and later Charpy, called attention to its constancy. In
four or five of the specimens dissected the last two tributaries
joined this duct at the same level, and in such a manner and
of such proportions as to appear to form a third pancreatic duct
traversing the caudal segment of the head parallel to the main
pancreatic duct. In no instances, however, was there any observ-

—

PANCREATIC DUCTS AND MINOR DUODENAL PAPILLA 207

able direct communication between these twigs and the duo-
denum.

The main duct drained the whole of the body, tail, and neck
of the gland, and in addition to these parts, in 66 per cent, or
fifty of the seventy-six specimens studied for this purpose, the
dorsal half of the head with nearly the whole of the caudal por-
tion of the ventral half (fig. 2). This restricted the accessory
duct to the ventro-cephalic portion in the immediate neighbor-
hood of the minor papilla and to a small portion of the ventro-
caudal segment. The main duct usually drained the whole of
the region of the head adjacent to the neck. In four specimens
the accessory duct drained the whole of the cephalic half of the
head. In three instances the main duct was restricted to the
dorsal and caudal portion of the head. In one other example
the accessory duct drained nearly the whole of the head of the
gland.

The outside diameter of the main duct, in those specimens
with a normal arrangement of ducts (88), taken a few millimeters
before it emptied into the duodenum and with the duct flattened
out, averaged 3.25 mm. The mean diameter was 3.0 mm. In the
body of the gland the duct averaged 3.0 mm. The smallest main
duct measured 1.5 mm. and the largest 4.5 mm.

There were three specimens in the series which presented a
rather unusual arrangement of ducts as represented in fig. 4.
In these instances the main duct, descending towards the right
into the caudal portion of the head, described a ‘loop,’ as shown in
the figure, before finally proceeding horizontally to the major
papilla. The accessory duct in these instances occupied its usual
ventral and cephalic position and joined the main duct before the
beginning of the ‘loop.’ In no instances was the main duct dup-
licated in the body of the pancreas as described by Bernard
nor was there found the spiral disposition of the pancreatic
duct as described by Hyrtl and figured in his Corrosion An-
atomie.

208 W. M. BALDWIN

Bile Duct

Accessory
Pancreatic Duct

Pancreatic Duct

Head ‘of Pancreas

Fig. 4 A rough; schematic sketch of the ventral surface of the head of the pan-
creas showing the typical arrangement of the pancreatic duct in those specimens in
which the ‘loop’ disposition prevailed.

3. The duodenal termination of the pancreatic duct in the major
papilla and its relation to the bile duct

Ever since Bidloo first noted the papilla common to both the
bile and the pancreatic duct, the relation of these two ducts to
each other in the ampulla has been the subject of considerable —
investigation. Bernard and Laguesse each mentioned one specimen
in which the main pancreatic duct opened into the duodenum apart
from the orifice of the bile duct. Bécourt also recorded another
instance. Schirmer reported twenty-two specimens (about 47 per
cent) among forty-seven investigated in which a mucosal fold sepa-
rated the orifices of the ducts in such a manner that a true ampulla
did not exist. Opie examined one hundred specimens. In eleven
instances no ampulla was present, the two ducts entering the duo-
denum separately. In the remaining cases the ampulla varied in
length from less than 1 mm. to 11 mm., while in only thirty speci-
mens did this measurement equal or exceed 5mm. The ampullary
orifice had an average diameter of 2.5 mm. Among twenty-one
specimens which Letulle studied in only six was there a true
ampulla.

PANCREATIC DUCTS AND MINOR DUODENAL PAPILLA 209

The main pancreatic duct in this series of one hundred speci-
mens approached the caudal aspect of the ductus choledochus to
fuse with its wall before penetrating the duodenal wall (fig. 3). In
two instances the main duct emptied into the caudal aspect of
the bile duct at 1.3 cm. and 0.7 em. respectively from the duo-
denal wall. Upon opening the ducts it was a noticeable fact that,
notwithstanding the apparent fusion of the walls outside of the
duodenum, the lumina did not unite until the duodenal wall had
been perforated.

Fig. 5 represents diagrammatically the two classes into which
the specimens reported and those studied in this investigation

--Bile Duct

Pancreatic
uct uct
Ampulla apilla
eptum
Lumen of_fiaags Lumen of
Duodenum ie

seem to fall. In A the walls of the two ducts are seen to fuse at
the level of the duodenal wall. The lumina, on the other hand, do
not fuse until the papilla has been entered. The thin mucous
septum is shown separating the two ducts for a distance of at
least one half of the papilla where the true ampulla can then be
said to begin. Fig. 5, B represents the other general appearance
noted, 7. e., complete isolation of the two ducts. The figure also
gives a fairly good representation of the foliated appearance of
the mucosa observed in the ampulla and mentioned at an earlier
date by Bernard.

210 W. M. BALDWIN

The distance from the mouth of the major papilla to the point
of junction of the two ducts in the ampulla averaged 4.8 mm.
(mean 4.0 mm.) in the ninety specimens dissected. In twenty
of the specimens (about 22 per cent), there could be found no
junction of the ducts, each opening side by side separately into
the duodenum through the major papilla. This appearance is
represented in fig. 5, B. A true ampulla was not present in these
cases. In two specimens the distance observed was0.5mm. In
twelve instances the partition was only 2.0 mm. from the mouth
of the ampulla.

In but one pancreas was the duodenal end of the main duct oc-
cluded (fig. 6). The duct in this instance was a mere impervious
twig which opened neither into the bile nor the accessory duct.
The accessory duct drained the whole gland.

4. The accessory pancreatic duct; course, tributaries, and drainage

The accessory duct was found to be present in each of seventy-
six specimens examined with that object in view. It waslocated
entirely within the substance of the cephalo-ventral segment of
the head (fig. 3), and pursued an arched course towards the duo-
denum. In no instance did it occupy a position wholly caudal to
the main duct.

Invariably the accessory duct lay upon a plane ventcal to that
of the main duct. Two curves were described in its passage to
the duodenum; the first of these, more pronounced and with its
concavity cephalad, occupied the duct end, while the other the
shorter of the two, was situated at the duodenal end with its con-
cavity looking caudad. This condition, present in forty-two (64 per
cent) of sixty-six specimens, is not clearly enough represented in
fig. 3. In twenty-one specimens (31 per cent) the duct described
a wide curvature with its concavity cephalad. Leaviag the main
duct it proceeded into the caudal portion of the head of the gland,
then, turning to pass ventral to the main duct, emptied into the
minor papilla. This appearance is represented in fig. 8. In the
three remaining specimens (5 per cent) the usual curvatures of the
duct were reversed, 7. e., a caudal concavity in the duct half with

PANCREATIC DUCTS AND MINOR DUODENAL PAPILLA 211

Bile Duct

Accessory
Pancreatic Duct

Main
Pancreatic Duct

Bile Duct

Accessory
Pancreatic Duct

Pancreatic Duct

Fig. 7 In this schematic sketch the ventral surface of the head of the pan-
creas is represented. Here the ducts are ‘inverted,’ 7. e., the accessory duct conveys
most of the drainage from the neck, body, and tail of the gland into the duodenum.
The main duct, occupying its usual caudal and dorsal position, is inferior in size
to the accessory duct but joins the bile duct to empty with it through the major
papilla into the duodenum.

Ae W. M. BALDWIN

a cephalic concavity at the duodenal end. Apart from the twenty-
one specimens above noted, in forty-five (69 per cent) the duct
was restricted to the cephalic and ventral segment of the head.

Charpy’s work agrees with the results of this investigation
regarding the part of the gland drained by the accessory duct.
Opie, however, thought that the accessory duct drained “the
anterior and lower part of the head’’ restricting for the main duct
a smaller mass of parenchyma ‘behind the larger lobe.’

In fifty-eight specimens (88 per cent) the duct approached the
duodenum with diminishing calibre; in six specimens (9 per cent)
the duodenal end was larger (fig. 7) while in two (3 per cent) both
ends were of the same size. These figures, as would be expected,
were compiled from those specimens in which the duct united with
the main duct in the usual manner, namely, from sixty-six speci-
mens. In ten other specimens where no demonstrable junction
was present, the accessory duct naturally approached the duode-
num with an augmenting calibre (figs. 9 and 10).

The outside diameter of the flattened accessory duct in these
sixty-six specimens, taken at the point where it perforated the
duodenal wall, averaged 1.2 mm. The smallest observed was 0.75
mm. and the largest 2.0 mm., with 1.0 mm. as the mean diameter
of this end of the duct. Under the same conditions the other end
of the accessory duct at its junction with the main duct measured
1.75 mm. with limits of 1.0 mm. minimum and 3.0 mm. maxi-
mum and with 1.5 mm. as the mean diameter.

In three other specimens, however, the maximum diameters
observed at the duodenal end were 2.5 mm., 3.0 mm., and 3.6mm.
respectively, but these were instances of inversion of the ducts,
7. é€., the main duct was inferior in size to the accessory duct as
represented in figs. 7 and 9.

These facts bear out Meckel’s statement that in the foetus
the two pancreatic ducts possess the same calibre, but as develop-
ment progresses the accessory duct undergoes a natural atrophy
at its duodenal end. This fact was also noted by Bernard and
verified still later by Schieffer upon five human fcetuses from 7.5
to 9 months of age.

PANCREATIC DUCTS AND MINOR DUODENAL PAPILLA 213

Accessory
Pancreatic Duct

8

Bile Duct

Accessory
Pancreatic Duct

Pancreatic Duct

=)

Fig. 8 This sketch represents the ventral surface of the head of the pancreas
showing the accessory duct passing through the caudal portion of the head.

Fig. 9 represents a condition present in five specimens of the seventy-six dis-
sected (6.5 per cent). The ducts do not unite. The accessory duct, larger than the
main duct, drains the whole of the body, tail, neck, and cephalic half of the head.
This is a persistence of the embryonic arrangement of ducts. To these ducts the
terms ductus pancreatis ventralis for the main duct and ductus pancreatis dorsalis
for the accessory duct are particularly applicable.

THE ANATOMICAL RECORD, VOL. 5, No. 5

214 W. M. BALDWIN

In ten of seventy-six specimens (13.2 per cent) the accessory
duct failed to jom with the main duct (figs. 9 and 10). The junc-
tion in the other sixty-six specimens, or 86.8 per cent of cases, was
found invariably in the head close to the neck of the gland (fig. 3).
Among these latter the accessory duct fused with the ventral sur-
face of the main duct in twenty-five (38.0 per cent), with the
caudal surface in twelve (19.0 per cent), the duct passing ventral
to the main duct; and with the cephalic surface in twenty-nine
specimens (43.0 per cent).

5. The minor papilla; relation to the accessory duct and micro-
scopical structure

The minor papilla was present in each of one hundred specimens
examined. As a means of studying more accurately the relation
of the accessory duct to the minor papilla, forty-six out of a
total series of fifty specimens were subjected to a microscopical
examination without first having been injected as a means of
ascertaining the condition of patency of the duodenal end of the
duct. A block of tissue comprising the papilla and the duodenal
wall with the adjacent pancreatic substance was imbedded in
paraffin, sectioned in series in thicknesses varying from 12 to 40u
and stained with hematoxylin and eosin.

Forty-one specimens (82 per cent) demonstrated a patent
accessory duct. In five (10 per cent) the duct was closed, ter-
minating blindly at the papilla. It seems needless to say that in
these last specimens the accessory duct communicated with the
main duct through an ample orifice. A feature especially worthy
of mention was the abrupt manner in which the accessory duct
in these five instances became constricted from an ample lumen
to one of capillary dimensions and then terminated abruptly at
the papilla. This abrupt dwarfing of the duct was no excep-
tional feature confined to these five isolated specimens. It was
the rule rather than the exception. In brief, as was frequently
verified, amplitude of calibre was no criterion of patency. In the
four remaining specimens of the series of fifty selected (8 per cent),
the patency of the accessory duct was so manifest as to be demon-

,

PANCREATIC DUCTS AND MINOR DUODENAL PAPILLA 215

strable upon gross dissection. In these, therefore, no microscopical
examination wasmade. ‘This gives, then, among fifty specimens
examined, a total of five (10 per cent) which did not communicate
with the duodenum. Further, in six other specimens the accessory
duct did not unite with the main duct, giving, therefore, a total
percentage of practical importance of eleven specimens out of
fifty (22 per cent) in which fluid could not pass from the main duct
into the duodenum through the accessory duct.

The shape of the papilla was uniformly rounded or conical with
a diameter averaging 2 mm. and an aperture quite variable,

Bile Duct

Accessory Duct

Main
Pancreatic Duct

Is

Fig. 10 shows an arrangement found in five specimens (6.5 per cent) of the sev-
enty-six dissected. The accessory duct is isolated and smaller than the main duct.
It drains but a small region in the immediate neighborhood of the minor
papilla, through which it opens into the duodenum.

most often not visible to the unaided eye. The epithelial cover-
ing did not differ in appearance from that found in the rest of the
duodenum (fig. 11, #). The mass of the papilla was composed of a
core (C.C.) This core, imbedded in the mucosa and submucosa
of the gut and extending obliquely from the muscularis (/) to
the epithelial covering of the papilla (2), consisted of a support-
ing framework of dense connective tissue, and appeared as a
constant factor in the structure of the papilla. It was present
when the accessory duct failed, indeed, its size, which contri-
buted largely to the proportions of the papilla, seemed less
referable to the presence of the accessory duct than to the

Fig. 11 (magnified 18 diameters) A longitudinal microscopical section of the
minor duodenal papilla showing the passage of the accessory duct through the core
of connective tissue.

PANCREATIC DUCTS AND MINOR DUODENAL PAPILLA 217

quantity of mucous glandular tissue enclosed within its stroma.
The prominent features, then, of the papilla were this core of
dense connective tissue containing many smooth muscle fibres
(M.F.) and enclosmg much mucous glandular tissue, with the
accessory duct (A.D.) traversing the middle of its substance. Its
whole appearance was strongly suggestive, however, of func-
tional regression remindful of Meckel’s observation regarding the
developmental atrophy of the duodenalend of the accessory duct.

The accessory duct (A.D.) passed directly from the pancreatic
tissue (P) of the head of the gland, which accompanied it up to the
duodenal wall, through both layers of muscular tissue (1).
Entering immediately into the substance of the core, it passed
through the middle of its stroma to open finally into the duode-
num. At the level of perforation of the duodenal wall, it under-
went an abrupt caudal bending. The angle of this flexure, as pre-
viously noted by Helly, varied from 20° to 30°. In thirty-seven
of the fifty specimens (74 per cent) the duct passed in a caudo-
ventral direction through the papilla; in six specimens (12 per
cent) it curved caudodorsally; and in the remaining seven
specimens (14 per cent) horizontally ventral. Occasionally it was
noted that the fibres of the muscularis formed a sphincter-like ring _
around the duct at the level where it perforated the duodenal wall.

There was no difficulty experienced in tracing the accessory
duct through the pancreatic tissue which accompanied it upto
the duodenal wall. The lumen was direct, uniform, and either
gradually enlarging or diminishing in calibre. Once that the mus-
cularis was perforated, however, the appearance of the duct was
transformed to a remarkable extent. The lumen now became
tortuous and irregular, dilating and narrowing, and, at times,
branching to reunite farther along in its course. To add to the
complexity of this arrangement the association with the duct in
the core of numerous mucous glands, whose individual or combined
ducts either opened directly into the accessory duct or independ-
ently into the intestine, rendered the tracing of the lumen of the
duct particularly difficult.

The amplitude of lumen of the accessory duct as it approached
the papilla offered no trustworthy suggestion of its condition of

218 W. M. BALDWIN

patency or-occlusion in the core. Oftentimes a duct with the
largest calibre dwarfed instantly to capillary dimensions upon
entering the core. On the other hand, occasionally the smaller
and most unpromising ducts traversed the core with a direct,
unsinuous, and even enlarging lumen. In six specimens (12 per
cent) the lumen of the accessory duct gradually increased in
size as it traversed the papilla towards the epithelial covering.
In ten specimens (20 per cent) the lumen sustained a pronounced
diminution in calibre, while in the remaining thirty-four cases (68
per cent) the duct was so tortuous and irregular as to make it im-
possible to say whether there was an actual increase or a reduc-
tion in size.

In the five instances in which the accessory duct did not com-
municate with the duodenum, the duct was found to have per-
forated the muscularis. In the core immediately adjacent to the
muscular coat, however, it suddenly underwent a diminution in
calibre and terminated blindly in the connective tissue of the
stroma of the core (C). In some of these specimens the strands
of connective tissue separating the duct lumen from that of the
adjacent mucous glands were so delicate that it seemed possible
and, indeed, quite probable that they could be broken down by
an injection mass even under the lightest pressure, thus giving
rise to erroneous conclusions as to the condition of patency of the
duct. Thus was confirmed both Henle’s and Helly’s objections
to injection methods.

In those instances where the duct was comparatively ample
and the lumen could be followed with little difficulty through the
papilla, the mucous glandular, and pancreatic tissues played only
subsidiary parts in the formation of the core and but little muscu-
lar tissue was discernible. In the majority of instances, however,
the duct, being constricted, formed but a small part of the core.
In these specimens the connective tissue and muscular stroma
were very prominent. In these, too, aside from the epithelial
lining of the lumen, there was no distinct wall to the duct. The
true wall ceased where the duct perforated the muscular coat of
the duodenum.

PANCREATIC DUCTS AND MINOR DUODENAL PAPILLA 219

The duodenal orifice in the instance of the small ducts appeared
like that of an intestinal gland, the lumen proper of the duct,
indeed, seemingly, opening into the fundus of the tubule and the
side walls not differing from those of the usua! intestinal gland.
Larger ducts, however, opened through what appeared to be
several fused tubules. This orifice occurred either upon the caudal
slope or upon the summit of the papilla, seldom upon its cephalic
aspect. '

Many glands (M.G.), which from general appearances seemed
to be mucous in character, were found associated with the acces-
sory duct. The characteristic staining of mucous glands could
not, however, be obtained in the instance of these glands owing,
doubtless, to their imperfect fixation. Kd6lliker noted the occur-
rence of these glands in the walls of the larger ducts of the pan-
creas apart from the papille.

These glands occurred in large, irregular, spherical groups
situated either with the accessory duct within the core or immedi-
ately outlying it in the loose connective tissue of the papilla. The
ducts of those glands situated within the core opened through irreg-
ular channels into the accessory duct. Those located near the
epithelial extremity of the core imbedded in the connective tissue
of the papilla opened directly upon the surface of the duodenal
mucosa, while those farther removed from the epithelium emptied
by longer channels, either into the accessory duct or upoa the
surface of the mucosa. The presence or absence of the accessory
duct did not seem to influence the number of these glands so
much as might be expected. In the five specimens of occlusion
of the duct, they opened either directly upon the surface of the
mucosa or indirectly through a lengthy, tortuous channel which
occupied the usual position of the accessory duct in the core.
When the duct was very large and patent the mucous glands were
fewer in number and much more scattered. No instances were
found where the glands were entirely absent.

In confirmation of Helly’s and of Opie’s earlier observations,
small masses of pancreatic tissue (P.7'.) were found in two situa-
tions, first, within the core close to the duodenal muscularis;
secondly, in the loose connective tissue of the papilla usually

220 W. M. BALDWIN

upon the caudal aspect of the core. This pancreatic tissue dif-
fered from the tissue of the pancreas itself only in the distribu-
tion of the supporting connective tissue, the latter occurring in
thick, well-marked septa isolating the lobules and acini from each
other. The ducts from the acini united into larger trunks which
emptied either directly into the accessory duct or independently
upon the epithelial surface of the duodenum.

The unstriped muscle tissue (V/.F.) contributed largely to the
thickness of the septa of the core. The fibres were scattered
either parallel to the long axis of the core or were disposed circu-
larly around some of the tubules. The amount of muscular
tissue was greater towards the muscularis side of the papilla
but few fibres reaching the level of the intestinal glands. There
could not be observed any relation between the condition of
patency or occlusion of the accessory duct and the number of these
fibres.

Claude Bernard thought that the papilla was contractile.

The relation of the muscularis mucose (M.M.) of the duodenal
wall to the tissue of the core could not be ascertained in every in-
stance. In some specimens it was continuous with the muscular
tissue of the core. In many others, however, it could be clearly
seen that there was no continuity of this structure with that in
the core.

The following pages present a tabular compilation of all of the
work which has been done upon the features presented in this
problem.

The minor papilla

PRESENT | ABSENT
Schirmer: 4.460), 2.5 ec oe eee 103 1
Helly: oes i eee 47 3
Charpy sicko aitt.cs os, eee eee 29 1
Vermeil. ac to ene ee 20 0
Baldwin.) 25.3520 eee eee 100 0

Thus it will be seen from the above that in about 98 per cent of
specimens the minor papilla is present.

PANCREATIC DUCTS AND MINOR DUODENAL PAPILLA 221

But four specimens of three papillze have been reported:

20 Sn ee 1
eT ss aes ss oo ee ee 1
2 LUGS rd hc 1
ivi pe. Se ee | 1

None of these accessory papillae have been studied microscopi-
cally. The papilla are of possible interest in two ways, first,
because of the occasional bifid character of the ventral anlage,
and, secondly, because of the occasional appearance of a third
duct in the caudal region of the head.

The accessory duct

PRESENT ABSENT

eter ee a e.....| 101 3
TL 29 1
Ul. 5.66 40h Bee ee 50 0
OCT ETT eS | 20 0
D2 tT TLE Se Sr ? 0
2D ae ? 0
EDT TOR Dryas be 50+ 0.
SST G0. gi a Wi 0
Oflu). ae eee 100 0
DLT er | 76 0

eRe SAPOV TiS ek thrill 443 4

Complete absence of the accessory duct according to these
figures seems to be a rare anomaly since it occurs in less than 1
per cent of specimens.

Condition of accessory duct at duodenal end. With microscopical method

PATENT CLOSED
Lota aco MRS A a rrr 40 10
DD a, a rrr 45 5

Zoe, W. M. BALDWIN

With injection method

GO Ghinmen: 2 720e0ss ho COE eee ee 85 | 19
CRATDY 5-4: sci. 0 eee ee er 9 21
Optes oie. bh erin de eee oe cee 79 21
Werneutls 220 oS re ee eee 20 0
Sappey: S:. --)4s qo HemenE te sees ee ree 16 1

Total :<2. gcse ake tee aie oe Een ee 209 62

According to the microscopical method, 15 per cent of ducts
are occluded; with the injection method about 23 per cent are
closed at the duodenal end.

Relation of main to accessory duct

JUNCTION NO JUNCTION

OPIGs ss: cuss oes 2 Soe Se ee eae 90 10
Diwan. 33 eo ee ee ? iL
15 (2) Neem ce cas sclera orsic WE etre dle: 48 2
CHAaTPY.. vcctor tere Oe oe oe eee 28 2
Schirmer’ oc sack hose cso hcbines © ee Oe eee 97 7
Vierneuil <... occ e aie eee eee eee 20 0
Baldwin... 245 5et eee eee 66 10

Total vo. yee ee ee 349 32

According to these figures a junction between the main duct
and the accessory duct is to be expected in over 90 per cent of
specimens.

The accessory duct is larger than the main duct

SPECIMENS EXAMINED INVERSION
Schitimer.c.apoicic os boop tee eee 104 3
Charpy. . 2.7. obds. 4 ooaan se eee ee | 30 on
BOrnar Geis cic ces cet eee ee eee 1
Morel and Duval......... EN ce ane Be | ? 1
Opiess.cc5. oe he ee eee ee 100 11
Biman... ccc ne tee eee ? 1
MOYS6 5.0... :0cs achaieeetaed ee ee ee ? 1
Baldwithsj.ni. 2 : Sees SR ee ee eee | 76 3

Total...:. 422 ee eee 310 24

,

PANCREATIC DUCTS AND MINOR DUODENAL PAPILLA aes

Distance between major and minor papillae

| NUMBER EXAMINED AVERAGE LIMIT
a a é i a 7 . cm cm.
Wermlardee tee. soe tc. re 2450 (2.0-4.0)
Schirmer 104 2.5-3:5
UGS RS 2Al 1.8 (1.0-3.5
Baidwanr i. we. sc ce Ss 100 2.0 (0.9-3 5)

6. The relation of the main pancreatic duct to the common bile duct
at the duodenal wall

Schirmer mentioned eleven instances among a series of forty-
seven specimens in which the pancreatic duct opened into the bile
duct and also fourteen instances in the same series in which the
bile duct opened into the pancreatic duct. In these cases the
single conjoined duct was the only one entering the ampulla of the
papilla. Verneuil seemed to believe that usually the pancreatic
duct received the bile duct and that, accordingly, the ampulla
of the papilla belonged to the pancreatic duct. The main duct
did not fail in any of the ninety specimens of my series, in one,
however, it was occluded at its duodenal end. Helly saw one
instance where the main duct was absent; Schirmer, four; Cru-—
veilhier, one; Charpy, one. (See topic 3, page 208.)

Occasionally the common bile duct opens into the duodenum
in company with the accessory duct. No such instance was found
in my series. Schirmer mentions five. Tiedemann mentions one
case where both pancreatic ducts emptied separately into the
duodenum apart from the common bile duct.

THE DUCTS JOIN TO

NO JUNCTION
FORM AN AMPULLA 2 SURSIGN

STS 3 OO ee ? 1
ILEOR BEE. bake soHe aoe Eee eee ? 1
SVCLNTIM EET e al 8 PES ARS a de 25 22
(URS: sos 83 6S) ARB oe 89 11
TORIES. 5 ce a 8 y S 9 12
IB DUGENTW. 2 oa le eae 70 20

224 W. M. BALDWIN

A B

Fig. 12 (dorsal view) represents the two conditions of the bile duct. In A
the duct passes through the tissue of the head of the pancreas. In B the duct
grooves the head of the gland but is not entirely surrounded by pancreatic tissue.

In about 25.8 per cent of specimens the ducts open separately
into the duodenum. In 74.2 per cent the ducts have a common
ampulla.

7. The bile duct and the major papilla

As an unavoidable adjunct to this study of the ducts of the
pancreas the relations of the terminal or pancreatic portion of the
bile duct were considered in this series of one hundred specimens.
The duct ran invariably caudally towards the median sur-
face of the second portion of the duodenum lying dorsal to the
head of the pancreas and producing a furrow upon that surface.
In no instance did it pass, as was observed by Helly, in a groove
between the duodenum and the pancreas. In 80 per cent of the
specimens the pancreatic tissue completely surrounded the duct
for a distance varying from 0.5 cm. to 5.0em. In 5 per cent of
specimens the duct received a partial investment without being
entirely enclosed by glandular tissue, while in the remaining 15 per
cent of specimens the bile duct grooved but was not covered by
the tissue of the head.

PANCREATIC DUCTS AND MINOR DUODENAL PAPILLA 225

The lumen of the bile duct underwent a marked contraction at
the duodenal wall before its junction with the main pancreatic
duct (fig. 5). Cephalad to this level a distinct bulging or ampulla
was noticeable. The difference in calibre between these two adja-
cent portions was less appreciable upon the external surface of the
duct than upon the internal. The outside diameter of the bile duct
at the level of its perforation of the duodenal wall was 5.4 mm.
in the one hundred specimens, that of the ampulla of the duct
averaged 6.4mm. The largest bile duct observed measured 15.0
mm. and the smallest 3:0 mm. This gives 6.0 mm. as the mean
diameter of this duct. These measurements are outside diam-
eters taken with the duct flattened out.

Letulle and Nattan-Larrier reported nineteen specimens in
which the common bile duct traversed the head of the gland, often
only a thin strip of pancreatic tissue separating the duct from the
duodenum. Usually the glandular tissue extended a distance of
only 2 or 4 cm. along the duct wall. The bile duct underwent a
diminution in size in the last centimeter averaging 8 to 9 mm.
in diameter. Several were from 12 to 14 mm.

O. Wyss found five specimens among twenty-two in which the
terminal portion of the common bile duct penetrated the head of
the gland. Helly studied forty specimens, in about half of which
the duct lay in a canal of pancreatic tissue.

Judging from the results of these investigations, we should expect
to find the terminal portion of the bile duct imbedded in pan-
creatic tissue in about 65 per cent of specimens.

Because of the nature of the material used in this investigation it
was found impossible to use the whole series of one hundred speci-
mens in the several portions of this problem. As many of them as
were suitable, were utilized, however, with the result that a smaller
number of specimens had to be reported upon in many of the essen-
tial features of the problem. This accounts, therefore, for the
somewhat confusing use of varying numbers of specimens.

In conclusion I wish to express my sincere appreciation of the
valuable advice and assistance given by Professor Gage, Dr. Kerr,
and by Dr. Kingsbury in the preparation of this paper and for
the numerous courtesies shown by their departments.

226 W. M. BALDWIN

BIBLIOGRAPHY

Batpwin, W. M. 1910 An adult human pancreas showing an embryological
condition. Anat. Rec., vol. 4, no. 1, pp. 21-22.

1910 A specimen of annular pancreas, Anat. Rec., vol. 4, no. 8, pp. -
299-304.

Duodenal diverticula in man. Anat. Rece., vol. 5, no. 3, pp. 121-141.
BarTHOLINUsS, TH. 1651 Anatomia reformata.
Bécourt, M. 1830 Recherches sur le pancréas.
BERNARD, Cu. 1856 Mémoire sur le pancréas.
Brpitoo, GovertT 1685 Anatomia humani corporis.
Bimar 1887 Conduits anormaux du pancréas. Gaz. hebdom. de Montpellier.

Buastus, G. 1677 Zootomia s. anatomia hominis.

BracHET 1897 Sur le développement du foie. Anat. Anzeiger. B. 13, N. 23,
S. 621-636.

Cuarpy, A. 1898 Variétés et anomalies des canaux pancréatiques. Journ. de
Anat. et de la Phys., p. 720.

CHOULANT, J. B. 1852 Geschichte der anatomischen Abbildungen.
CRUVEILHIER, J. 1833 Traité d’anatomie descriptive.

Feirx, W. 1892 Zur Leber und Pancreas Entwickelung. Arch. f. Anat.
GEGENBAUR, ©. 1890 Anatomie des Menschen.

DE GRAAF, REGNER 1671 Tractatus anat. med.

von Hatuer, A. 1764 Elementa physiologiae corp. hum.

HamBurGER 1892 Zur Entwickelung der Bauchspeicheldriisen. Anat. Anzeiger.

Heuiy, K. 1898 Beitrage zur Anatomie des Pankreas und seiner Ausfiihrungs-
ginge. Arch. f. mikr. Anat.

1901 Zur Pankreasentwickelung der Séugethiere. Arch. f. mikr.
Anat. u. Entwickelungsgesch.

1904 Zur Frage der primiren Lagebeziehungen bei der Pankreasanlagen
des Menschen. Arch. f. mikr. Anat.

HENLE, J. 1866 Eingeweidelehre.
Hits, W 1885 Anatomie menschlicher Embryonen. Bd. 3, Leipzig.

Huscuxe 1845 Traité de splanchnologie et des organs des sens. Traduction de
Jourdan. Paris.

PANCREATIC DUCTS AND MINOR DUODENAL PAPILLA 227

Hyrtt, J. 1873 Die Corrosions—Anatomie und ihre Ergebnisse. Wien.

Incatts, N. W. 1907 Beschreibung eines menschlichen Embryos von 4.9 Mon.
Archiv. fiir mikros. Anat. u. Entwickelungesch. 70, 506-576.

JANKELOWITz, A. 1895 Ein junger menschlicher Embryo und die Entwicklung
des Pankreas bei demselben. Arch. f. mikr. Anat.

JANOSIK 1895 Le Pancréas et la Rate. Bibliogr. anatom.

JouBIn 1895 Développement des canaux pancréatiques. Th. de Lille.
Ko.iMann, J. 1897 Handatlas der Entwickelungsgeschichte des Menschen.
KG6LLIKER 1889 Handbuch der Gewebelehre des Menschen, 6 Aufl.
LacuEsse 1894 Le pancréas apres les travaux recents. Journ. de |’ Anat.
LetTuLtLte 1898 Arch. des Sciences médicales.

Mayer 1819 Journal complémentaire des Sciences médicales, t. 3, p. 283.

Mecket 1812-1816 Handbuch der path. Anatomie und Anatomie comparée,
tid:

Mitne-Epwarps 1860 Legons sur la Physiologie et l’Anatomie comparées.
Moret et Duvat 1883 Manuel de l’anatomiste.

von Moratt, J. 1677 Vademecum anatomicum...:

Natran-LARRIER 1898 Bull. Soc. Anat., Paris.

NorunaGeL 1898 Handbuch der speciellen Pathologie und Therapie. (Oser.
Pankreas)

Orie, E.L. 1903 Anatomy of the Pancreas. Johns Hopkins Hospital Bulletin,
vol. 14.

Disease of the Pancreas, Its Cause and Nature. 1903. Second
edition, 1910.

Puisatix 1888 Etude d’un embryon humain de 10 mm. Arch. de Zoolog. Exp.
et Générale.

Rrioutanvs, J. 1649 Opera anatomica, p. 811.
ROLLESTIN AND Fenton 1900-1 Jour. Anat. Physiol., vol. 35, p. 110.

Santorini, J.D. 1775 Anatomici summi septemdecim tabulae quas edit. Michael
Girardi.

Sappey 1873 Traité d’anatomie.
ScHIEFFER 1894 Du Pancréas dans la série animale. Th. de Montpellier.

Scuirmer, A. M. 1893 Beitrag zur Geschichte und Anatomie des Pankreas.
Inaugural Dissertation. Basel.

228 WwW. M. BALDWIN

Sross 1891 Zur Entwickelungsgeschichte des Pancreas. Anat. Anzeiger.

Swaen, A. 1897 Recherches sur le développement du foie, du tube digestif, de
l’arriére-cavité du péritoine et du mésentére. Jour. de |’Anat. et de la
Physiol.

SwaLtweE 1668 Pancreas pancrene adornante.

Tuyna, F. W. 1908 Models of the pancreas in embryos of the Pig, rabbit, cat, and
man. Am. Jour. Anat., vol. 7, no. 4.

TIEDEMANN 1819 Journal complémentaire des Sciences médicales, t. 4, p. 330.
TIEDEMANN AND GMELIN 1826 Verdauung nach Versuchen.

VERNEUIL 1851 Mémoire sur quelques points de l’anatomie du pancréas. Gaz.
médic. de Paris.

VESLING, J. 1647 u. 1666 Syntagma anatomicum.

Vouixer, O. 1903. Uber die Verlagerung des dorsalen Pankreas beim menschen.
Arch. f. mikr. Anat.

Wetscutivus, L.C. 1698 Tabulae anatomicae.

Winstow 1732 Exposition anatomique.

Wirstina, C. 1642 Figura ductus cuiusdam....

Wyss, O. 1866 Zur Aetiologie des Stauungsikterus. Virchow’s Archiv.

ZIMMERMANN 1889 Rekonstruktionen eines menschlichen Embryos von 7mm.
Anat. Anz.

Reprinted from THe ANnatomicat ReEcorp, Vou. 6, No. 3 =
March, 1912

A METHOD OF FURNISHING A CONTINUOUS SUPPLY
OF NEW MEDIUM TO A TISSUE CULTURE
IN VITRO’

MONTROSE T. BURROWS

From the Anatomical Department, Cornell Universtty Medical College, New York

ONE FIGURE

The method devised by Harrison for the cultivation of tissues
in vitro has demonstrated that many varieties of tissues may
survive and grow for a period of time outside the animal organ-
ism. Harrison originally devised this method for studying con-
ditions surrounding the early differentiation of the nerve fiber and
for this purpose it has answered admirably as it probably will for
the differentiation of many other tissues. During the last year and
a half this method with its modifications has been used by many
workers as a means of studying the effects of salts and various
animal extracts and fluids upon growth. The experiments of
this kind have shown that absolute and constant differences,
expressed in terms of rate and extent of growth are not obtained |
when identical physical conditions surround in each case the
growing tissue unless the media used in these experiments are of
widely different composition and contain substances which act
promptly on the cell (cytotoxines, ete.). Such a result is to be
expected in cultures where the period of great activity is imme-
diate and endures for only a short period of time. ‘The tissue
vitality is undoubtedly sufficient to allow a considerable activity
even in a medium which would ultimately bring about cellular
death. Again, the chemical composition of the medium neces-
sarily changes continuously throughout the period of cellular
activity. In such cultures no sustained rate of growth is to be
expected even if an ideal medium be employed. It was neces-
sary, therefore, to improve the technic so that growth might

1 Read before the American Association, of Anatomists, December 27, 1911, at
Princeton, N. J.
141

142 MONTROSE T. BURROWS

be continued over a longer period and render the growth more
nearly comparable to cellular activities in the animal body. In
this way problems of cellular metabolism and problems dealing
with tissue growth and differentiation in various media may be
attacked.

The first step toward such improvements has resulted in a
methcd whichsupplis the tissue continuously with a known quan-
tity of new media and at the same time removes the waste pro-
ducts without in any way disturbing the growing cells. The
fresh medium is carried by means of a cotton wick from a reser-
voir at one end of a slide through a culture chamber and into a
receiving reservoir situated at a lower level at the opposite end
of the shde (fig. 1). In the culture chamber the wick is teased
apart into its individual fibers which adhere to the surface of the
cover glass and thus simulate a capillary system. The tissue is
placed in this open network of cotton fibers and held there by a
drop of coagulated plasma. The culture medium passes slowly
along the wick through the culture and collects in the receiving
chamber. The medium about the tissue is continuously changed
by this means.

The supplying chamber (fig. 1, a) is blown from glass. It con-
sists of two compartments, the horizontal or reservoir, and the
vertical or wick chamber. These two chambers are connected
by a glass tube, which comes from the bottom of the reservoir up
and over to enter the upper end of the wick chamber through a
small capillary point. The reservoir is open to the outside by a
long vertical tube. The wick chamber is connected with the cul-
ture chamber (fig. 1, b) by two tubes. One tube carries the wick;
the other acts as an air tube which equalizes the pressure on the
two ends of the wick.

The rim of the culture chamber b, is made of cork. The cen-
tral cavity of the cork is closed by a cover glass above and a long
glass slide below. The tubes of the supplying chamber and re-
ceiving chamber enter through holes cut horizontally through this
cork rim (see figure). All parts are carefully sealed together with
paraffin. The wick coming from the supplying chamber is spread
out on the under surface of the cover glass and here the culture is
planted among the cotton fibrils.

SUPPLY OF NEW MEDIUM TO A TISSUE CULTURE 143

The receiving chamber, fig. 1, c, is made of glass, and consists of
a horizontal reservoir situated below the level of the slide. One
tube connects this reservoir with the culture chamber and another
tube opens to the exterior as shown in the figure. The wick passes
from the cover glass through the glass tube to end above the sur-
face of the liquid in the receiving chamber. The wick does not
completely fill the tube. The arrangement allows free air com-
munication between the receiving chamber and the culture cham-
ber. The receiving chamber receives air by its communication

Fig. 1 Complete culture apparatus with rubber tube which connects with air
pressure; a, supplying chamber; b, culture chamber; c, receiving chamber.
with the exterior through the vertical tube which is plugged with
loose sterile cotton.

Blocks of wood held together with bolts are fastened by fric-
tion rigidly to the glass slide. A stand for the apparatus and firm
supports for the glass chambers are thus formed. The entire
apparatus is compact and strong and the contained culture may
be examined under the microscope.

THE ANATOMICAL RECORD, VOL. 6, NO. 3

144 MONTROSE T. BURROWS

The.glass chambers with the wick inserted are sterilized in the
autoclave. The cork is sterilized in hot paraffin. The-cover glass
and the slide are sterilized by dry heat. To set up the apparatus
the cork is removed from the hot paraffin and drained, the cham-
ber tubes are inserted into it and the cover glass sealed over its
surface. The wick is teased apart on the inner surface of the cover
glass and the tissue planted. The apparatus is then immediately
placed on the slide and all connections sealed with paraffin, and
the chambers are now fastened to the wooden supports.

The reservoir of the supplying chamber is filled with the liquid
medium and its open tube is plugged with dry sterile cotton.
The open end of this vertical glass tube is connected with the
pressure apparatus. The fluid is driven at a constant rate by air
pressure from the reservoir into the wick chamber.

To refill or empty the chambers the vertical tubes are flammed,
the cotton removed and the fluid entered or removed by sterile
pipettes. Freshly sterilized cotton plugs are inserted and the
apparatus again connected with the air pressure. Great care
must always be taken to keep all parts of the apparatus and the
media free from bacterial contamination.

Growth in such an apparatus has been tested with embryonic
chick tissues in a medium of blood serum prepared from adult
chickens. Growth of such tissues is vigorous and can be main-
tained for a considerable period of time. Hearts of embryo chicks
and small pieces of heart muscle can be kept beating with great
regularity. Small bits of ventricle beat actively until all muscle
fibers have wandered apart and are lost in dense connective tissue
outgrowths.

With this apparatus I have been able to test the effects of
various media on the growing cells during any period of their
activity. The growth is first established in control media. The
media to be tested is then added and its effects recorded. In
this manner the effects of many media may be tested on the grow-
ing cells without in any way disturbing or changing their physical
surroundings. Changes in the chemical composition of the media
can be tested by a comparison of the original media with samples
obtained from the receiving chamber.

[From THn JoHNsS Hopxins HospiraL BULLETIN, Vol. XXII, No. 242,
May, 1911.]

THE RELATIONSHIP BETWEEN THE NORMAL AND
PATHOLOGICAL THYROID GLAND OF FISH.

By J. F. GuDERNATSCH.

(From the Department of Anatomy, Cornell University Medical
College, New York City.)

Recent investigations of the thyroid gland of Teleosts have [152]
revealed a great many facts that may be of value to the com-
parative pathologist. The thyroid gland of these fish attracts
at present a good deal of attention in cancer research, since
it is often liable to cancerous degeneration, especially in
artificially reared trout and salmon.

The normal anatomy of the gland was briefly described in a
paper read before the meeting of the American Association of
Cancer Research, November 27, 1909. * It was especially em-
phasized that one of the most striking features of the thyroid
in bony fish is the absence of a connective tissue capsule, such
as exists in other vertebrates. Later this fact was again
pointed out by Marine and Lenhart. It will readily be seen
that the absence of a capsule makes it rather difficult to
define the normal extension of the gland. It would perhaps
be better not to use the term “thyroid gland” at all in this
group of animals, since physiologically isopotent units (fol-
licles) are not so arranged as to form a closed organ, but are
distributed over a wide area (Fig. 2 and Plate 1). This
distribution varies not only with the species, but also with the
individual, and is dependent entirely on mechanical influences,
mainly pressure from the sides of the surrounding tissues,
and pull enacted by the growing connective tissue fibers and [153]
blood and lymph vessels. The latter force works chiefly in the

* An extensive analysis of the normal conditions of the Teleost
thyroid will be found in Jour. of Morphology, V. 21, Suppl., 1911.

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[153] early development of the gland, when thyroid cells are carried
off from the main point of growth to distant regions, where
they form new centers of multiplication.

The thyroid gland develops around the stem of the ventral
aorta (Fig. 1), in many species the main bulk lying between
the branches to the first and second gill arches. This locality
is filled with connective tissue and fat, and is enclosed dorsally
by cartilages or bones and ventrally by muscles. Thus the
region available for the thyroid tissue is rather limited, and
therefore the follicles tend to fill every space that is offered
by the surrounding structures. Not uncommonly follicles are
found far from the center of thyroid development, invading
muscles (Plate 1, C) or creeping into the crevices which exist

{152]

yj
H
4

Fic. 1—These diagrammatic drawings show the expansion of
the thyroid gland in the three species: Oncorhynchus, Salvelinus
and Opsanus. J, aortic bifurcation; JZ, IJJ and IV, the second,
third and fourth branchial arteries.

between the osseous lamelle of the bones in this region (Fig.
2 and Plate 1, A).

The thyroid gland of the Teleosts is thus a rather indefinite
organ in its shape, having the tendency to lose its unity and
break into numerous small parts. In some species, the trout
and others, this tendency manifests itself most strikingly, so
that thyroid follicles are found even far out in the gill arches
along the gill filaments (Plate 1, D).

The spreading apart of the thyroid follicles over a wide
area and the invasion of neighboring tissues are a normal
feature and of no pathological significance. By such an in-

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vasion the surrounding structures are not destroyed. Often [153]
the term “invasion” is even incorrect. Thus Marine and
Lenhart’s statement that the normal follicles invade the bones

*is not appropriate, since they do not invade true bone or car-
tilage tissue, but merely the spaces that are present between
the osseous or cartilaginous lamelle (Fig. 2 and Plate 1, A).
In their paper Fig. 6 demonstrates this fact definitely, al-
though it is supposed to show a true invasion. On the other

[154]

Fig. 2—Section through the thyroid gland of Brevoortia, an-
terior to the aortic bifurcation. AJ, first branchial arteries.

hand, Gaylord was able to show specimens in which thyroid (154)
tissue, belonging to a diseased gland, had actually invaded or
infiltrated true cartilaginous tissue. The latter invasion, of
course, is never seen in the anatomy of the normal gland, but

is a strictly pathological feature. Whether or not it is due to

a cancerous growth of the gland, may still be an open question.
Strong evidence seems, however, to point in that direction.

(3)

[153]

PLATE I.—A and B, sections through the thyroid gland of @

bifurcation; B, near the second branchial arteries, the region
of Salvelinus. C, in the aortic bifurcation; D, near the secon:

(Thyroid follicles in all figures shown in solid black. Traiy
parts stippled. Arteries in heavy lines. Veins in light lines)

branchial arteries.)

[153]

est extension.
ial arteries.

2 uscles lined. Longitudinal muscles in polygons.
‘Mh sinuses in broken lines.

C and D, sections through the thyroid gland

Skeletal
A, ventral aorta, AI and AIJ,

1154) Should the so-called thyroid carcinoma of brook trout be
a cancerous growth and not a mere hyperplasia, as Marine and
Lenhart believe, then the question of metastasis again demands
the pathologist to keep in mind the lack of a capsule. Cer-
tainly no detached nodules in or around the gill region can
safely be called secondary tumors, since such misplaced struc-
tures in all probability are merely parts of the primarily dis-
eased gland. However, tumors on the tip of the jaw or around
the anus, as Gaylord has found them, can hardly be explained
as due to normally misplaced thyroid particles. Whether they
are true secondary growths or simply implantations, further
experimental-investigations may show.

Histologically the thyroid gland of the Teleosts offers a
great many interesting peculiarities. The size of the follicles
varies between very wide limits. Aside from the fact that in
young embryos it is naturally very small, the size of follicles
cannot be taken as a reliable indication of the age of the fish.
It seems much more probable that it is a sign of the age of the
individual follicle. »

The follicular epithelium varies from an almost flat to a
very high, columnar type. It is different in each individual
and may somewhat depend on the age and the physiological
condition of the animal. Yet the type of the epithelium is
not always uniform in all the follicles, sometimes very marked
differences are found (Fig. 3). Even in the individual follicle
the height of the epithelium may vary, probably due to differ-
ent pressure from outside. It was now and then observed,
that in oblong follicles the epithelium on the two longer sides
would be lower than on the shorter ones.

The colloid material is sometimes present in all the fol-
licles, in other glands it may appear in some only, in still
others it may be entirely lacking. It, again, is no definite
sign of the age of the animal, although it may be somewhat
dependent on the age of the fish, its sex (egg-carrying females,
for instance) and other inherent factors that need further
investigation. The colloid certainly is a sign of the physio-
logical state of the individual follicle, yet we do not under-
stand it well enough to interpret our observations in a defi-

(6)

nite manner. There can be no doubt that all the follicles ofa [154]
gland are not in the same state of physiological activity.
Otherwise it cannot be explained why (Hiirthle’s) colloid-
forming cells appear in some follicles only, sometimes in a
group of neighboring follicles, so that we can easily distinguish
“colloid zones ” from non-colloid-forming parts of the gland.

In interpreting their results after iodin treatment of the
thyroid gland of the pike Marine and Lenhart lay great stress
on the histological appearance of the treated and not treated

a

f } , ‘ . . ‘ * > 2 Z i Uy /

v ' ‘i NY. 4 > a AL Py 4
si) A —— ns Z SS, 7) YY
@: SW 2 BRR Sa ~ i 4

_ Fic. 3.—Section through the thyroid gland of Salvelinus fon-
ao the different heights of the follicular epithelium.
ae t= B

glands. Yet from the above discussion it seems obvious that
the anatomy of the gland as well as its histology, as far as
the type of epithelium and the colloid formation are con-
cerned, makes it rather difficult for the microscopist to dis-
tinguish a normal from a hyperplastic, and the latter in turn
from a “reverted ” thyroid gland in these fish.

The presence or absence of the colloid material has no

(7)

[154] significance whatsoever. If all glands, the follicles of which

[155

eS)

spread out far from the main bulk even into the gill region,
are highly hyperplastic, then, according to Marine and Len-
hart, the colloid material should be nearly or entirely absent
in‘them. Yet it is present throughout the gland.

The type of the epithelium is also a perfectly unreliable
guide in regarding a gland as hyperplastic. When, of course,
the epithelium shows marked foldings and protuberances into
the lumen, the hyperplastic condition is evident.

Further studies on the carcinoma of the fish thyroid will
have to take into account the peculiar anatomy and histology
of this organ in the Teleosts. Many conditions which might
be regarded as pathological may prove to be normal as soon
as our knowledge of all the factors involved is sufficiently
broadened.

BIBLIOGRAPHY.

Gaylord, H. R.: An Epidemic of Carcinoma of the Thyroid
Gland Among Fish. J. Am. Med. Ass., LIV, 227, 1910.

Gudernatsch, J. F.: The Structure, Distribution and Variation
of the Thyroid Gland in Fish. J. Am. Med. Ass., LIV, 227, 1910.

Gudernatsch, J. F.: The Thyroid Gland of the Teleosts. J. of
Morphology, XXI, 709, 1911.

Marine, D., and Lenhart, C. H.: On the Occurrence of Goitre
(Active Thyroid Hyperplasia) in Fish. Johns Hopkins Hosp.
Bully, XOXO byl S10:

Marine, D., and Lenhart, C. H.: Observations and Experiments
on the so-called Thyroid Carcinoma cf Brook Trout (Salvelinus
fontinalis) and Its Relation to Ordinary Goitre. J. Exper. Med.,
SGuie owlity ale)il().

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Reprinted from THE AMERICAN JOURNAL OF ANATOMY, VOL. 11, No. 3
March, 1911.

HERMAPHRODITISMUS VERUS IN MAN

J. F. GUDERNATSCH

From the Department of Embryology, Cornell University Medical College,
New York City

SEVEN FIGURES
THREE PLATES

Hermaphroditismus verus is of such rare occurrence and so
eminently important in our knowledge of the development of
the genital organs, that it would seem worth while to add a new
case to the few so far recorded. Numerous instances of supposedly
true hermaphroditism have been described, but only in rare
instances have they stood a critical consideration. ;

The microscopic diagnosis in many cases has been incorrect,
particularly in cases where the normal structure of the tissues had
been altered by neoplasms so that their identification was almost
impossible. Some instances of true hermaphroditism have been
reported in which histological examination was neglected; yet with-
out a microscopic investigation the correct interpretation of
malformations of this kind is at least doubtful.

The ‘ovotestis’ to be described in this article was taken from
an individual forty years old who came to the hospital to be oper-
ated upon for tumor of the right inguinal region. In the left
inguinal canal a similar, but somewhat smaller nodule was
detectable.

The external genitals were of the female type; labia majora
and minora were well developed and an introitus vagine was
present. The noticeably peculiar feature was the extremely
enlarged clitoris with the opening of the urethra on its ventral

THE AMERICAN JOURNAL OF ANATOMY, VOL. 11, No. 3

267

268 J. F. GUDERNATSCH

side, so that the organ offered rather the aspect of a hypospadic
penis than that of a clitoris.

No uterus was present and the vagina ended blindly (atresia
vagine). During the course of embryonic development, there-
fore, the greater part of the Millerian ducts had been lost and the
formation of the genital apparatus must have inclined toward the
male type. A prostate-like organ was felt attached to the ure-
thra, yet its real nature remains doubtful, since no microscopic
examination could be made. In other cases in which both a vagina
and prostate have been found they were seen to be connected with
one another, but in the present instance no detectable communi-
cation existed between the vagina and the supposed prostate.

The distribution of the hair on the body was that typical of
the female. The pelvis was wide, but mammary glands were not
developed, and the larynx was externally that of the male. The
secondary sexual characters were not so decidedly of the male
type that there arose any doubt about the sex of the individual,
although the knowledge of the malformation existed. The indi-
vidual was believed by herself and her associates to be a woman.

The individual has never menstruated, sexual intercourse has
never taken place, and libido sexualis is not present. Her psy-
chic disposition is thatof a woman and she earns a living asacook.

The tumor in the left inguinal region was left in place since it
did not cause inconvenience and that in the right channel was
removed. The nodule extirpated had about the form of a testicle
with attached epididymis. It measured 6 em. in length and 5
em. in width and thickness. Histologically it proved to be male
genital tissue, with the exception of a small nodule which exhibits
a structure very similar to that of an ovary. Dr. James Ewing
diagnosed this region of the specimen as true ovarian tissue.
Others who were asked to examine the sections agreed with Dr.
Ewing in considering the tissue ovarian.! ;

1 T wish to thank Prof. A. Kohn, the well known histologist in the German Uni-
versity of Prague, for his careful examination of the sections and for the many sug-
gestions he made in regard to their interpretation. The preparations were also
- demonstrated before the Eighth International Zodlogical Congress in Graz, and
the structure was interpreted by all as ovarian.

HERMAPHRODITISMUS VERUS IN MAN 269
THE TESTICULAR STRUCTURE

The mass of the testicle is surrounded by an extremely broad
tunica albuginea, the elements of which are arranged in a some-
what undulating manner and contain elastic fibres with but few
nuclei and vessel. As might be expected the testicular tissue
proper (fig. 2) is not of the appearance of the normal male sexual
gland since the organ developed under very abnormal conditions.
It resembles the well known pathological condition of a degenerat-
ing testicle, and the hyaline type of degeneration is typical of the
kryptorchic mammalian testicle retained in the inguinal canal.
The sections through the contorted tubules vary much in size, the
smaller ones are circular in shape, the larger ones more or less
oval and some a little bent or even S-shaped owing to the plane of
section. The average diameter of the tubuli contorti is much
smaller than normal. The epithelium which lines the tubules
shows only one row of basal cells, and these are propably all of the
Sertoli’s cell type being rather large, triangular or cubical in shape
with clear cytoplasm and large nuclei (fig. 2, cf). From their sur-
faces protoplasmic processes project into the lumen. Germ cells
seem to be entirely absent since there are no indications of sperma-
togonia, spermatids or spermatozoa. It is not improbable that in
the younger years of the individual anlagen of germ cells were
present in the tubules, but failed to undergo further development
on account of the abnormal physiological conditions. It must be
assumed that formerly germ cells existed, for without them the
development of male sexual glands is hardly conceivable.

The cells of Sertoli show different stages of degeneration as is
indicated by the different densities and staining abilities of the
nuclei. In some cells the nuclei are shrunken and lie in a nuclear
cavity. This degeneration of the epithelial cells is probably due
to the above mentioned hyaline degeneration of the wall of the
seminal tubules. The inner layers of the tunica propria upon
which the epithelial cells lie are swollen, while the constituent
cell nuclei disappear entirely, forming a hyaline mass which lies
as a band between the follicular wall and the epithelium (fig. 2
ct). This swollen band of cells pushes the epithelium towards the

270 J. F. GUDERNATSCH

lumen which thus becomes more and more occluded and often
with gradual dissolution of the cells becomes entirely obliterated.
The hyaline band exhibits a somewhat fibre-like structure with
processes from it extending between the epithelial cells. It varies
in thickness and in some places is entirely missing, and along with
this variation in thickness the degeneration of the epithelial cells
presents a regional distribution. In some regions the epithelial
cell nuclei stain in a normal manner and are situated towards the
periphery, in other regions the cell limits are indistinct and the
nuclei lie near the lumen. This accords with former observations,
and Finotti states that in the kryptorchic testicle, even when the
individual is not a hermaphrodite and the germinal epithelium in
places develops spermatozoa, the degeneration is not of uniform
degree in all regions.

Wherever there is a membrana propria left in the form of a
thin layer of spindle-like connective tissue cells, elastic fibres are
usually present.

The testicle under consideration offers still another peculiarity.
The interstitial tissue is enormously increased so that the seminal
tubules are in places pushed far apart (fig. 2, 2). Connective
tissue fibres are comparatively scarce in this tissue.

The interstitial cells are normal in appearance, the majority
being of a triangular or polygonal shape. The large nucleus
(there are occasionally two nuclei) possesses one or more nucleoli
and is usually excentrically situated. The cytoplasm is somewhat
denser than that of ordinary connective tissue cells, it is finely
granular and often contains in some regions a very fine brown
pigment. Of the so-called Reinke’s crystals nothing could be
detected.

The interstitial cells are usually arranged either in small
irregular groups or narrow streaks, often, however, they are united
in large compact nests and grow so excessively that they actually
invade the tunica albuginea. There is no relationship between
these cells and the blood vessels as is often claimed.

This striking increase in the number of interstitial cells is a
feature well known to the human embryologist as well as to the
pathologist. During the fourth month of embryonal develop-

HERMAPHRODITISMUS VERUS IN MAN 271

ment these cells constitute about two-thirds of the parenchyma
of the testicle. Their number is also very much increasetl in the
kryptorchie testicle. Whether the large amount of interstitial
tissue in the present case is an embryonal condition due to arrested
development, or simply a secondary pathological feature, is
difficult to say. The latter, however, seems to be more probable
since there are no indications, except perhaps the entire lack of
germinal epithelium, that the gland did not develop normally.
It probably degenerated later on account of its unusual position.
Finotti claims that the gland does not degenerate for sucha
reason, but on account of an early predisposition to do so.

The entire accessory system of the male genital gland, rete

testis, ductuli efferentes, ductus epididymis and vas deferens,
are present. The globus major is, as far as the arrangement of the
efferent ducts is concerned, rather well developed. The epithe-
lium, however, is very degenerate in places (fig. 4), though to-
wards the duct of the epididymis it approaches a normal condi-
tion. The epithelium in some tubules is of the low cubical type
without foldings (fig. 3), while in others it shows the normal pro-
jections into the lumen with alternating columnar and cubical
cells.
The structure of the epididymis resembles in parts that of the
epodphoron and since both organs are derived from the Wolffian
body, it is not impossible that in a true hermaphrodite we might.
find them somewhat mixed.

The ductus epididymidis is normally developed. The muscular
coat of the efferent ducts increases in thickness as they approach
the epididymis. There are numerous elastic fibres among the mus-
cle cells; if these, as Stéhr states, do not appear until puberty is
reached we must conclude that the entire efferent system reached
a mature state independently of the testicle. This is also empha-
sized by the fact that the better developed parts are those
farthest removed from the testicle. The duct of the epididymis,
for instance, has a normal, highly columnar, ciliated epithelium,
which shows no signs of degeneration. In the lumen is seen cell
detritus, a finely granular mass and numerous concrements. The
muscular coat of the spermatic cord is much increased.

272 J. F. GUDERNATSCH

The sclerotic blood vessels, as everywhere seen in the sections,
are typical of the kryptorchic testicle.

THE OVARIAN STRUCTURE

The female portion of the genital gland is rather small, the rudi-
mentary ovary being a little nodule only 3 mm. in length and 2
mm. in width and thickness. It is enclosed within a cyst in the
tunica between the testicle and the head of the epididymis (fig.
1, 0). The typical ovarian stroma is easily recognized by the
arrangement of the spindle-shaped connective tissue cells (fig.
5). This structure is nowhere else to be found in the human body.
Cortical and medullary portions are distinguishable. The former
consists of dense connective tissue rich in cells, and traversed by
small blood vessels. The slender cells sometimes resembling
smooth muscle fibres, are arranged either in strands or twirls. In
the central portion of the ovarian body the connective tissue con-
tains fewer nuclei and its elements are arranged in broader
streaks. The blood vessels are large and bent.

The entire nodule is surrounded by a single-layered, cubical
or cylindrical epithelium (fig. 5) which although rather primi-
tive shows here and there slight cellular differentiation. Some
cells are larger and broader and their nuclei are large and
more circular and contain less chromatin than the neighboring
cylindrical cells (figs. 6, 7, p). These cells are very probably
primordial ova, yet a definite diagnosis cannot be made. How-
ever the decision that the body is ovarian in structure is sufficiently
warranted by the typical stroma with its surface epithelium.

The ovary remained in an early stage of development as is
indicated by the rather high columnar cells in certain regions
(fig. 6). A migration of primordial ova into the stroma and the
formation of Graafian follicles has not taken place, probably
due to the abnormal conditions of development. This accords
with earlier reports on the subject which state that in all cases
of hermaphroditism, whether true or spurious feminine, the epi-
thelial part of the ovary is below the normal in development.
Various transitions have been described from almost mature

HERMAPHRODITISMUS VERUS IN MAN ies

ovaries to those containing only primordial follicles or eyen empty
follicles. ,

The primitive and rather small female portion found in this
hermaphroditic genital gland indicates perhaps that in many
cases of spurious hermaphroditism traces of ovarian tissue might
be found, provided the entire testicular tissue be thoroughly
searched, so far, however, this has never been done.

That both types of germinal tissue are in a hypoplastic condi-
tion is explained by Halban in the following way, the impulse
for development, which normally is concentrated upon one system,
in cases of hermaphroditism iscalled to act upon two systems and
thus is insufficient to force either to the normal degree of develop-
ment.

In the present case male and female tissues are found in close
contact in one gland, but an intermixing of the two kinds of tissue
does not exist, and therefore the term “‘ovotestis,’’ as is often used
for this kind of gland, does not seem to be appropriate. In the
true ovotestis of invertebrates male and female sexual cells
are produced by one glandular structure.

The male part of an ‘ovotestis’ is as arule considerably larger
and further developed than the female. Kopsch and Szymono-
vicz have observed this in all hermaphroditic vertebrates and
it likewise holds for the present case. This individual, however,
shows many more external female characters than one should
expect when considering the large male part of the genital gland.
If the interstitial cells of the testis are really responsible for the
accessory sexual characters then the person in question should
show a typical male condition. The body of the individual is
not a perfect woman, yet the male characters are not so out-
spoken that she could be called a man-woman. This fact is sur-
prising from the study of the genital gland tissue, as far as this
could be investigated, but it must be remembered that the
actual amount of ovarian tissue the individual really carried in its
body is not known. As has been mentioned above, anodule existed
in the left inguinal canal similar to that described from the right,
this in all probability may have also been genital gland tissue and
it might have contained a preponderance of ovarian material.

274 J. F. GUDERNATSCH

Such is not improbable since in malformations of this kind a great
difference between left and right genital glands has often been
observed. Salén, for instance, describes a case of true hermaphro-
ditism, in which the right side contained an ‘‘ovotestis,”” while
on the left a perfect ovarium was found. Lilienfeld states that in
all cases of disturbed development of the genital region the female
type is predominant on the left side. In this imdividual a
large ovary may have been present on one or both sides higher
up in the abdominal cavity, or there may have been more ovarian
tissue present during an earlier period of life than can now be
detected. This latter suggestion would account for the develop-
ment of the strong female characters of the individual. Ker-
mauner states in Schwalbe’s ‘‘Die Missbildungen des Menschen
und der Tiere” that ‘“‘whether the entire defect may be regarded
as primary aplasia, or later involution, cannot always be decided.
In no case can it be entirely denied that microscopic remnants of
ovarian tissue, perhaps transformed beyond recognition, may be
located somewhere in the abdominal cavity.”

Whatever circumstances were responsible for the strong female
characters of this person, it is interesting that along with them the
male sexual apparatus developed to the perfection here described.
The cavities and the concrements of the epididymis would indi-
cate that a secretory function was performed by the epithelium,

This case of true hermaphroditism recalls the old theory of
Waldeyer according to which there is a bisexual anlage of the geni-
tal gland, as opposed to Lenhossek’s idea of an indifferent anlage.
Instances in which male and female genital tissue are found
next to each other speak at least for Waldeyer’s view that the
ovary develops from a different region of the genital ridge from that
of the testicle even though they may not entirely support his
theory of hermaphroditism. In all the cases of true hermaphro-
ditism the ovary occupies the same relative position to the testicle.
It seems strange that there should always be a sharp distinction
between the two kinds of tissue and never an undefined mixing
of both elements (true ovotestis) as might be expected, if all cells
of the germinal epithelium could produce either male or female
tissue.

HERMAPHRODITISMUS VERUS IN MAN aes

Every embryo has the anlagen of the efferent ducts for the
expulsion of both male and female products of the genital glands,
which indicates that the male and female sexual apparatus are
rather distinct from one another, thus it does not seem impossible
that two distinct regions of the germinal epithelium might exist
next to one another, one giving off the male, the other the female
primordial cells. Why is the Miillerian duct always laid down in
the male if there are no female tendencies in the undifferentiated
embryo? It seems that in every embryo there is a trace of a
female tendency. Some authors, Benda for instance, go so far
as to claim “that the primary anlage of the entire sexual system
of the vertebrates must be regarded as female.’’ Waldeyer’s
view, however, may be correct, that the Miillerian duct alone is
the primary efferent duct for the genital glands in both sexes. He
believes that in its function it corresponds to the primitive open-
ing for the products of the gonads in the lowest vertebrates, the
porus abdominalis. This scarcely seems possible, since in some
fish adbominal pores and efferent genital ducts exist side by side
and thus the second do not replace the first. Benda on the basis of
Waldeyer’s view may be justified in his conclusion that at no
time do both ducts, Wolffian and Miillerian, exist as parallel
genital ducts. Yet this is not entirely true, for the Wolffian duct,
even in early periods, when it certainly serves as the mesonephric_
duct, must possess the potential faculty of developing into a male
genital duct. This faculty is possessed by the duct in all embryos,
whether the further development is male or female.

In the resulting female the possession of the quality to develop
the mesonephric duct into a male sperm duct seems likely, if not
proven, by the fact that the remnants of the Wolffian body, the
Epooéphoron and Parodphoron, closely resembling the structure of
some parts of the epididymis, are found to exist.

The present case is interesting in still another direction. The
sister of this hermaphrodite also shows irregularities in the forma-
tion of the external genital organs. Unfortunately only the
testimony of laymen could be secured regarding this. In mana
hereditary tendency towards hermaphroditism has never been
scientifically proven, though several cases have been supported

276 J. F. GUDERNATSCH

by laymen. Reuter, however, found among three pigs of one
litter one true and two spurious hermaphrodites, and in a later
litter from the same sow a pseudo-hermaphrodite occurred.

From this investigation the sex of the individual remains unde-
termined. According to Virchow it is an ‘individuum utriusque
generis.’ According to Klebs the condition is a typical herma-
phroditismus verus. Klebs regards an individual as a true her-
maphrodite, when the genital glands of both sexes are united in it.
The physiological state is of no importance, simply the anatomical
fact. An anatomical hermaphroditism seems to be all we can
expect to find in vertebrates. The physiological hermaphroditism,
as is normally the case in invertebrates, may hardly be looked for
inman. In the higher vertebrates the persistence of both genital
glands, is looked upon as an imperfect development and under such
circumstances it is only natural that their physiological faculty
should be reduced.

Our knowledge of the etiology of these malformations is almost
nil, since in general our conceptions of the principles involved in
the development of sex are still rather vague. It remains for the
anatomist and embryologist, perhaps for the experimenter, to
bring about a deeper understanding of these abnormalities.
So long as the interest in them is reserved for pathologists and
clinicians, and the malformations of the external genitals remain
the only thing of interest, the literature regarding such anomalies
will be as Benda states ‘“‘overcrowded with sensational reports,”’
with no exact investigation of the anatomical and histological
features.

Hermaphroditism in the sense that separate testicles and ova-
ries are found has not been demonstrated in man, nor even in other
mammals beyond doubt. Yet there are four cases of the so-
called ovotestis on record, two of these with neoplastic changes
in the male portion. The present is therefore the fifth recorded
case of true hermaphroditism in man.

HERMAPHRODITISMUS VERUS IN MAN ae

LITERATURE CITED ;

Brenpa, C. 1895 Hermaphroditismus und Missbildungen mit Verwischung des
Geschlechtscharakters. Ergebn, d. allg. Path., vol. 2, p. 627.

Born, G. 1894 Die Entwickelung der Geschlechtsdriisen. Erg. An. u. Entw.,
vol. 4, p. 592.

Corsy, H. 1905 Removal of a tumor from a hermaphrodite. Brit. Med. J.,
vol. 2, p. 710.

FisicEr, J. 1905 Beitrige zur Kenntnis des weiblichen Scheinzwittertums.
Virchow’s Arch. f. path. An., vol. 181, p. 1.

Finotti, E. 1897 Zur Pathologie und Therapie des Leistenhodens nebst einigen
Bemerkungen iiber die grossen Zwischenzellen des Hodens. Arch. f.
klin. Chir., vol. 55, p. 120.

Hasan, J. 1903 Die Entstehung der Geschlechtscharaktere. Arch. f. Gynaek.
vol. 70, p. 205.

Hansemann, D. 1895 Uber die grossen Zwischenzellen des Hodens. Virchow’s
Arch. f. path. Anat., vol. 142, p. 538.

HirscHrewp, M. 1905 Ein Fall von irrtiimlicher Geschlechtsbestimmung. Monat-
Schr. 1. Harnkr. u. sex. Hyg., vo.. 2, p. 53.

1905 Ein seltener Fall von He:maphroditismus. Monatsschr. f. Harnkr.
&. sex. Hyg., vol. 2, p. 202.

HoFMEISTER 1872 Untersuchungen iiber die Zwischensubstanz im Hoden der
Saugetiere. Sitzungsb. Akad. Wiss. Wien, vol. 65.

JaNosik, J. 1887 Bemerkungen iiber die Entwicklung des Genitalsystems.
Sitzungsber. Akad. Wiss. Wien, vol. 99, 3. Abt., p. 260.

Kopscu, Fr. u. SzymMonowrez, L. 1896 Ein Fall von Hermaphroditismus verus
bilateralis beim Schweine, nebst Bemerkungen iiber die Entstehung
der Geschlechtsdriisen aus dem Keimepithel. An. Anz., vol. 12, p. 129.

Luxscu, F. 1900 Uber einen neuen Fall von weit entwickeltem Hermaphro-
ditismus spurius masculinus internus. Ztschr. f. Heilk., Abt. f. Path.,
vol. 21, p. 215.

Merxner, K. 1905 Zur Frage des Hermaphroditismus verus. Ztschr. f. Heilk.,
Abt. f. prakt. Anat., vol. 26, p. 318.

Mimaukowicz, G. 1885 Untersuchungen tiber die Entwicklung des Harn- und
Geschlechtsapparates bei Amnioten. Intern. Monatsschr. f. An. u.
Phys: vol. 2, p. 1.

NEUGEBAUER, F. 1908 Hermaphroditismus beim Menschen. Leipzig.

Puruipps, J. 1887 Four cases of spurious hermaphroditism in one family. Trans-
act. Obst. Soc. London, vol 28, p. 158.

278 J. F. GUDERNATSCH

Pick, L. 1905 Uber Adenome der mannlichen und weiblichen Keimdriise bei Her-
maphroditismus verus und spurius. Berl. klin. Woch., vol. 43, p. 502.

1905 Uber Neubildungen am Genitale bei Zwittern. Arch. f. Gynaek.,
vol. 765 p: LOL.

Puato, J. 1897 Die interstitiellen Zellen des Hodens und ihre physiologische
Bedeutung. Arch. f. mikr. Anat., vol. 48, p. 281.

REINKE, Fr. 1896 Beitrige zur Histologie des Menschen. Arch. f. mikr. Anat.,
1896, vol. 47, p. 34.

Reizenstetn, A 1905 Uber Pseudohermaphroditismus masculinus. Miinchn.
med. Woch., vol. 52, p. 1517.

Satby, E. 1899 Ein Fall von Hermaphroditismus verus unilateralis beim Men-
schen. Verh. Deutsch. Path. Ges., vol. 2, p. 241.

ScuickELE, G. 1906° Adenoma tubulare ovarii (testiculare). Hegar’s Beitr. z.
Geburtsh. u. Gynaek., vol. 11, p. 263.

ScHwaLBE, EK. 1906 Die Morphologie der Missbildungen des Menschen und der
Tiere, 6. Jena.

Simon, W. Hermaphroditismus verus. Virchows’ Arch. f. path. An., vol. 172,
0) th

Spanaaro, 8. 1900 Uber die histologischen Verainderungen des Hodens und des
Samenleiters von Geburt an bis zum Greisenalter. Anat. Hefte, vol. 18.

Tournevux, F. 1904 Hermaphroditisme de la glande génitale chex la taupe
femelle adulte et localisation des cellules interstitielles dans le segment
spermatique. Comp. rend. de l’assoc. des anat., Toulouse, p. 49.

Uncer, E. 1905 Beitrag zur Lehre vom Hermaphroditismus. Berl. klin. Woch.,
vol. 42, p. 499.

Wa.upEYeEerR, W. 1870 Eierstock und Ei. Leipzig.

4

PLATE 1
EXPLANATION OF FIGURES
1 General view of the relative positions of testicle, ¢, ovary, 0; and epididymis,
a IDrey ileibe

2 Testicular tissue, showing the hyaline wall of the convoluted tubules, ct;
and the large masses of interstitial cells, 7. Dia. 1:90.

PLATE 2
EXPLANATION OF FIGURES

3and4 Sections through different parts of the epididymis. The tissue in fig. 3
resembles somewhat a parovarian structure. Dia. 1:90.

PLATE 3
EXPLANATION OF FIGURES
5 Ovarian tissue. Dia. 1:50.

6 and 7 Germinal epithelium of the ovary; p, somewhat differentiated cells,
probably primordial ova. Dia. 1:200.

PLATE 1

ME RMAPHRODITISMUS VERUS IN MAN

I’. GuDERNATSCH

J.

9

PLATE

HERMAPHRODITISMUS VERUS IN MAN

J. F. GupERNATSCH

THE AMERICAN JOURNAL OF ANATOMY, VOL, 11, No. 3

HERMAPHRODITISMUS VERUS IN MAN PLATE 3 ~

J. F. Guppernatscn aay

THE AMERICAN JOURNAL OF ANATOMY, VOL. 11, NO. 3

Abdruck aus den
Verhandlungen des VIII. Internationalen Zoologen-Kongresses zu
Graz vom 15.—20. August 1910. ’

Ein Fall von Hermaphroditismus verus hominis’).
Von J. F. Gudernatsch (New York City).

Eine 40 Jahre alte Frau war wegen eines Tumors in der
rechten Leistengegend, der ihr seit einiger Zeit Beschwerden ver-
ursacht hatte, auf die chirurgische Klinik gebracht worden. Bei
Betastung konnte an der bezeichneten Stelle eine etwa hiihnerei-
groBe Geschwulst, in der linken Gegend eine dhnliche, etwas
- kleinere, ermittelt werden.

Die duBeren Genitalien boten das Bild des Weiblichen dar.
Labia majora und minora waren gut ausgebildet, ein introitus
vaginae vorhanden. Auffallig war nur die tibermaBig groBe Aus-
bildung der Clitoris und die Ausmiindung der Harnréhre an der
Unterseite des Clitorisschaftes, so daB man eher das Bild eines
hypospadischen Penis vor sich hatte. Bei innerer Untersuchung
der Genitalregion wurde dagegen konstatiert, daB kein Uterus
vorhanden war, sondern die Vagina blind endigte; wohl aber ist
ein prostata-ahnlicher Korper tastbar. Es ist somit der gréBte
Teil der Mitillerschen Gadnge verloren gegangen, die Ausbil-
dung der Genitalorgane hat sich also in der fiir das mannliche
Geschlecht geltenden Richtung bewegt. Ob ibrigens der der
Harnrohre anlagernde Koérper Prostata war, ist sehr fraglich, da
die histologische Diagnose fehlt ; denn eigentlich miiBten ja Scheide
und Prostata miteinander in Zusammenhang stehen.

Weiterer Befund: Die Behaarung des Mons veneris weiblich,
sonst am Ké6rper keine Behaarung, das Becken breit, Milch-
driisen nicht entwickelt, Kehlkopf eher mannlich, im Gesicht ein
leichter Bartanflug, wie er etwa bei Frauen nach der Menopause
auftritt, Kopfhaar lang. Der Gesamthabitus und die Bildung
der auBeren Geschlechtsorgane sind nicht so mannlich, daB iiber
das Geschlecht des Individuums Zweifel aufgetaucht waren. Es
wurde von sich selbst und seiner Umgebung fiir ein Weib ge-
halten.

1) Die ausfithrliche Arbeit ist erschienen in ,,The American Journal of
Anatomy, Vol. XI.‘

Vierte Sitzung der zweiten und dritten Sektion.

Jt
~I
—

Die Kranke hat niemals menstruiert, Beischlafversuche haben
keine stattgefunden, Libido war nie vorhanden. Die Psyche ist
weiblich; Patientin verdient als Kéchin ihren Lebensunterhalt.

Die Geschwulst der linken Inguinalgegend wurde vorlaufig
belassén, da sie keine Beschwerden verursachte, die der rechten
wurde entfernt. Das exstirpierte Gebilde hatte annahernd die
Form eines Hodens mit Nebenhoden. DaB man es mit einem
solchen tatsachlich zu tun habe, bestatigte die histologische Dia-
gnose. Die Frau wurde deshalb als mannlicher Scheinzwitter
bezeichnet. Erst als mehr Schnitte aus einer ganz zufallig ge-
wahlten zweiten Partie des Tumors angefertigt wurden, fand man
ein kleines Gebilde, das unter dem Mikroskop eine der des Ova-
riums sehr ahnliche Struktur erkennen lheB. Wegen der Selten-
heit eines derartigen Befundes und seiner groBen entwicklungs-
geschichtlichen Bedeutung wurden die Schnitte auch mehreren
Fachleuten, unter anderen Herrn Professor Alfred Kohn
in Prag, zur Begutachtung vorgelegt. Die Struktur wurde als
unzweifelhaft ovariell bezeichnet.

Schnitte durch die Hodensubstanz zeigen kein normales Ge-
webe, was ja erklarlich ist, da die Keimdriise unter ganz ab-
normalen .Verhaltnissen zur Entwicklung gelangt ist. Sie bietet
das jedem Pathologen wohlbekannte Bild des degenerierenden
Hodens dar, und zwar ist diese Art der Degeneration, die hyaline,
fiir den im Leistenkanal retinierten, kryptorchischen Saugerhoden
typisch. Man sieht Durchschnitte durch die gewundenen Samen-
kanalchen in Menge, aber von dem dieselben unter normalen
Verhaltnissen auskleidenden Epithel findet sich nur eine Reihe
von basalen, wohl ausschlieBlich Sertolischen Zellen. Der
Hode begann also zuerst eine normale Entwicklung, erreichte
eine verhaltnismaBig hohe Ausbildung und ist erst spater ent-
artet. Die hyaline Degeneration der Membrana propria und die
Degeneration des Samenkanalchenepithels ist tibrigens nicht an
allen Stellen gleich weit vorgeschritten. Letzterer Befund steht
im Einklang mit fritheren Angaben. So hat schon Finotti
gezeigt, daB beim Leistenhoden nicht immer alle Partien gleich-
maBig degenerieren.

Noch eine andere Eigentiimlichkeit zeigen die Schnitte durch
den Hoden. Das Hodenzwischengewebe ist auBerordentlich
machtig entwickelt, so zwar, daB die interstitiellen Zellen oft zu
ganzen Nestern vereinigt sind. Auch das ist eine typische Er-
scheinung, einerseits beim etwa vier Monate alten Fétus, wo diese
Zellen nach Nagel zwei Drittel des Parenchyms ausmachen,
andererseits beim kryptorchischen, degenerierenden Hoden. Wel-
cher von den beiden Fallen hier zutrifft, ob der Reichtum an
Zwischengewebe ein embryonaler Zustand ist oder ein sekundar
pathologischer, ist schwer zu entscheiden. Das letztere hat die
groBere Wahrscheinlichkeit fiir sich; denn es ergibt sich kein

Gudernatsch, Ein Fall von Hermaphroditismus verus hominis. 572
Grund gegen die Annahme, daB sich der Hode anfangs normal
entwickelt hat und erst spater infolge der ungewohnlichen Lage-
rungsverhaltnisse degeneriert ist.

Auch die sklerotischen GefaBe, wie sie in den Schnitten zu
sehen sind, sind fiir den im Leistenkanal retinierten Hoden typisch.

Die tibrigen Teile des Hodens, Rete, Epididymis, Ausfiih-
rungsgange, sind vorhanden, alle natiirlich mehr oder weniger
pathologisch verandert. Nebenhoden ist ziemlich typisch gebaut,
ahnelt aber gleichzeitig auch sehr der Struktur des Parovariums.
Beide Gebilde sind ja Abk6émmlinge des W olffschen Korpers
und als solche homologe Organe.

Den weiblichen Anteil am Aufbau der Keimdriise stellt ein
sehr kleines, etwa bohnenfOrmiges Knoétchen dar. Am Schnitt
kann man an der Anordnung der spindelformigen Bindegewebs-
zellen sofort das typische ovarielle Stroma erkennen, wie es sonst
in keinem Organ des Korpers gefunden wird. Man sieht genau
so wie in der Rindensubstanz des normalen Ovariums kleinere
GefaBe in einem dichten, kernreichen Bindegewebe, dessen meist
schlanke Elemente hie und da eine wirbelartige Anordnung er-
kennen lassen und teilweise glatten Muskelzellen ahneln. In der
zentralen Partie des Gebildes haben wir ahnlich wie im Mark des
Ovariums groBe, gewundene GefaBe; das Bindegewebe ist viel
armer an Kernen als in der Rindenschicht und in gréberen Lagen
angeordnet. Das Gebilde ist an seiner Oberflache von einem
einreihigen Epithel bekleidet, wie wir es vom Ovarium her kennen.
Bei naherer Durchsicht der Schnitte bemerkt man hie und da
eine Differenzierung in diesem Epithel; einzelne Zellen sind etwas
groBer und breiter als die ihnen benachbarten Zylinderzellen,
sehen etwas gequollen aus, und die Kerne in ihnen sind armer
an Chromatin als die in den tibrigen Zellen. Man diirfte kaum
fehl gehen, diese Zellen als Anlagen von Ureiern anzusehen. —
Die Diagnose ,,ovarielles Gebilde“ 1aBt sich schon durch den
typischen Bau des Stromas mit dem umgebenden Epithel be-
grinden. Allerdings muB gesagt werden, daB das Organ auf
einer auBerst friihen Entwicklungsstufe stehen geblieben ist, wo-
rauf auch die hohen Zylinderzellen des Keimepithels hindeuten.
Zur Ausbildung eines Follikelapparates, Eimwandern von Ur-
eiern in das Stroma und Anlage von Graafschen Follikeln
ist es nicht mehr gekommen. Dies stimmt wiederum vdllig mit
den bisherigen Befunden auf diesem Gebiete iiberein. Es sind
alle bisher bekannten Falle von Hermaphroditismus, seien sie
echte oder unechte, dadurch ausgezeichnet, da der epitheliale
Teil des Ovariums immer hinter der Norm, gewoéhnlich stark,
zuriickbleibt, so daB von nahezu reifen Ovarien bis zu solchen,
die nur Primarfollikel, ja selbst leere Follikel zeigten, alle Uber-
gange gefunden wurden.

Aus den anatomischen und histologischen Befunden geht her-

573 Vierte Sitzung der zweiten und dritten Sektion.
vor, daB der mannliche Anteil am Aufbau dieses Ovotestis be-
deutend gréBer und auch in der Entwicklung weiter vorgeschritten
ist als der weibliche. Es soll tbrigens, wie Kopsch und
Szymonowicz feststellten, auch bei niederen Sdugetieren,
falls ein Ovotestis sich findet, der ovarielle Anteil immer kleiner
sein als der testikulare. Und doch sind in unserem Falle die se-
kundaren Sexualcharaktere durchaus nicht absolut nach der
mannlichen Seite ausschlaggebend, obwohl man bei dem groBen
Reichtum an interstitiellem Gewebe gerade das Gegenteil er-
warten sollte. Die Patientin ist zwar kein absolutes Weib, doch
sind die mannlichen Anzeichen nicht einmal so stark, daB man
sie als Mannweib bezeichnen konnte. Sie gilt ja iibrigens fiir ihre
Umgebung als Weib. Dieser Umstand scheint mit den Befunden
an der Keimdriise in Widerspruch zu stehen. Doch wissen wir
leider nicht, wieviel ovarielles Gewebe die Frau tatsdchlich in
ihrem K6rper besitzt. Wie eingangs erwahnt, findet sich bei der
Patientin in der linken Inguinalgegend ein ahnlich knotiges Ge-
bilde, wie das aus der rechten entfernte. Es ist mit groéBter Wahr-
scheinlichkeit anzunehmen, daB es sich auch dort um Keimgewebe
handelt, der Charakter desselben aber lieBe sich nur durch eine ge-
naue histologische Untersuchung feststellen. Daf tiefgreifende
Unterschiede zwischen rechter und linker Keimdriise bestehen,
wurde schon 6fters beobachtet. So fand sich z. B. bei dem Fall
von Hermaphroditismus verus, den Salén_beschreibt, linker-
seits ein vollstandiges Ovarium, rechts eine Zwitterdrise. Es
soll nach Lilienfeld itiberhaupt bei menschlichen Zwittern
auf der linken Seite das weibliche Geschlecht tberwiegen. Es
kann aber auch anfanglich mehr weibliches Keimgewebe vorhanden
gewesen sein als spater gefunden werden kann; ,,denn“, sagt
Kermauner in Schwalbes ,,Die Mi8bildungen des Menschen
und der Tiere“, ,,ob der vollstandige Defekt auf primare Aplasie
oder nachtragliche Involution zuriickzufihren ist, ist nicht immer
zu entscheiden; in keinem Falle kann man es ausschlieBen, daB
mikroskopische Reste des Ovariums, vielleicht zur Unkenntlich-
keit verandert, irgendwo an der Bauchwand liegen geblieben sind”.

In dem hier beschriebenen Falle scheint es unwahrscheinlich,
daB der Knoten, der auf der linken Seite getastet werden kann,
nicht auch zum gréBten Teile mannliches Keimgewebe enthalt ;
denn es diirfte ja ein Descensus versucht worden sein. Unbedingt
aber mu die Frau ihrem, wenn auch nicht ausgesprochen, so
doch ziemlich stark weiblichen Typus nach zu schlieBen, viel
mehr weibliches Keimgewebe in sich haben, als wir in dem kleinen
Knoten auf der rechten Seite gefunden haben. Es ist sehr wahr-
scheinlich, daB auf der einen oder der anderen oder auf beiden
Seiten sich neben dem Leistenhoden noch Ovarien héher oben in
der Bauchhohle vorfinden. Das sind natiirlich MutmaSungen,
die nur durch eine genaue Autopsie und mikroskopische Dia-

Gudernatsch, Ein Fall von Hermaphroditismus verus hominis. 574

gnose auf ihre Richtigkeit gepriift werden kénnen. Sicher laBt
sich nur sagen, daB die Person viel weibliche Charaktere aufweist,
und es erscheint dann interessant, daB trotz dieses Umstandes
der mannliche Keimapparat sich zu einer derartigen Hohe ent-
wickeln konnte, wie die Praparate es zeigen. Es ist sogar, wie
man aus den Hohlraumen und Konkrementen im Nebenhoden
schlieBen muB, unbedingt zu einer sekretorischen Tatigkeit ge-
kommen.

Der Fall ruft die Anschauung Waldeyers von der ur-
spriinglich zweigeschlechtlichen Anlage des Keimbezirkes gegen-
iiber der neueren Lenhosseks von der anfangs indifferenten
ins Gedachtnis zurtick. Beide Anlagen stehen, man konnte sagen,
im labilen Gleichgewicht zueinander, bis die eine aus unbekannten
Griinden die Oberhand gewinnt und die andere dann unterdrtckt
wird. In diesem Falle ware eben die schwachere Anlage doch
noch bis zu einem gewissen Grade zum Durchbruch gekommen.

Noch in einer anderen Hinsicht ist der demonstrierte Fall
interessant. Die Schwester dieses Hermaphroditen zeigt namlich
ebenfalls UnregelmaBigkeiten im Bau der auBeren Geschlechts-
organe. Fir den Menschen ist bisher Hereditat oder verwandt-
schaftliche Beziehung beim Hermaphroditismus noch nicht wissen-
schaftlich einwandfrei nachgewiesen worden, wohl aber fand
Reuter bei einem Wurf von drei Schweinen einen echten und
zwei Pseudohermaphroditen, und in einem spateren Wurf des-
selben Mutterschweines wiederum einen Pseudohermaphroditen.

Ich bin geneigt, die Patientin als echten Zwitter anzusehen
und sttitze mich dabei auf die Einteilung von Klebs, der
von einem echten Zwitter spricht, wenn die Keimdriisen beider
Geschlechter in einem Individuum angelegt sind. Die physiolo-
gische Leistungsfahigkeit kommt dabei nicht in Betracht. Herma-
phroditismus in dem Sinne, daB Hoden und Eierstécke beider-
seits getrennt vorkommen, ist beim Menschen noch nicht ge-
funden worden. Hingegen sind vier Falle von sogenanntem Ovo-
testis — zwei allerdings mit neoplastischer Veranderung des
mannlichen Anteiles — unbestreitbar festgestellt worden, denen
sich der vorliegende Fall als fiinfter anschlieBt.

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{Reprinted from BroLocicaL BULLETIN, Vol. XXII., No 3 February, 1912.)

THE OSMOTIC AND SURFACE TENSION PHENOMENA
OF LIVING ELEMENTS AND THEIR PHYSIO-
LOGICAL SIGNIFICANCE!

J. F. McCLENDON.

CONTENTS.
1, InaTerac live atet@inh.cerctoncto Sac OOD GO AOTC 6 SS Hie a a Poe II4
EEO smOLicsE Menomena TE ANCS rc. siaise aeitiess dicis acs cicis cases vspedleed cle 120
MMe Edio-elecubi Cue WeHOMEHA cay, a5 stelssJeacse oe cons ces ba aec.cceneeeeus 127
&. 1 PERU . SSB 6 od ap Aclae SEO r ae Skt ae ras re 127
Zam B VIN era GuINGLVGsesrois akcie fait ¢ x< Hew ous «cc's ee eys/s baa cents Sere I29
eee N EEL OR ECT Oy tit OUT Ea cer cy erate vanas rato ici<' ors cree vieis 2.40 eee w le ¥ Slers 134
4. The Propagation of the Bio-electric Changes..................2.. 136
uP IN EC OSI Pte Pe a ei eet eP CPA ofc ete eo cle cre cie Gas gus cie'e wa wd side 139
V. Osmotic Properties of the Blood Corpuscles.................02ece0. 142
WA AD SOnpUOUIanN Cl: SECEELIONs sre en spis aac 6 6 aleve clactacs sev ecuisiivciv owes 148
Ti, JANSSON DOE) (etree ee sto Cee OGG GO Oe eee eee 148
2. Osmotic Relation of Aquatic Animals to the Medium............. 149.
2% Secretion of He vrrp uate ISSUEMPUICE? gris ss «s.r fe:5 Sk ecls.e.cie vw oeeie ox 152
4, [EEO 6 oe GS oS dd BOOB OHO ADEs Ono ee ee es 153
WIL, (Gail IDIsSHOW. Soe. Gae Cfo nO bee a On ar 154
PREFACE.

This paper formed the basis for two lectures given before the
class in physiology at Woods Hole, July 7 and 8, 1911, although
owing to limited time, some parts were omitted. Since then
there has appeared a second edition of Héber’s ‘‘ Physikalische
Chemie der Zelle und Gewebe,’’ which reviews much of the
literature considered in this paper. However, owing to an
entirely different mode of presentation, it is hoped that the
present treatment of the subject might be helpful to many
general readers, some of whom would not read Héber’s book.

1From the Embryological Laboratory of Cornell University Medical College,
New York City.
Fis

114 J. F. MCCLENDON.

I am indebted to several persons for suggestions, especially
to Dr. Ralph Lillie! and Professor B. M. Duggar.

I. INTRODUCTION.

‘

The object of this paper is to bring the ‘“‘vital’’ phenomena,
as far as possible, within the scope of physics and chemistry, and
not to elucidate physical and chemical processes. It should
therefore be borne in mind that the osmotic phenomena of
‘“‘dead’’ systems are not all satisfactorily explained.

The Vant Hoff-Arrhenius theory of osmosis concerns itself
with the number of particles, molecules and ions, in solution,
and is applicable to dilute solutions, in which the total volume of
the dissolved particles is negligible. However, in more con-
centrated solutions, the volume of the dissolved particles is of
the same importance as the volume of the molecules in gases, as
expressed in Van der Waal’s equation. Also the dissolved
particles bind molecules of the solvent and so reduce the volume
of the free solvent. :

That the molecules and ions of a dissolved substance bind
some molecules of the solvent, follows from the work of Jones
and his collaborators.2, Compare also the work of Pickering.*
Jones concludes that the larger the number of molecules of water
of crystallization, the greater the hydrating power of a substance
in aqueous solution. The number of molecules of water bound
by one molecule of the solute usually increases with dilution up
to a certain point (the boundary between concentrated and
dilute solutions, beyond which there is no heat of dilution).
The bond between ions and the solvent is also indicated by the
phenomenon known as “electrical transference.’ If an elec-
trolyte and a non-electrolyte be dissolved in water and an
electric current passed through the solution, water will be carried
along with the ions to the electrodes.

With these corrections, the Vant Hoff-Arrhenius theory
accounts for osmotic pressure, but does not show why many
substances exert no osmotic pressure, in other words, why no

1 Cf. this journal, 1909, XVII., 188.
* “ Hydrates in Aqueous Solution,’’ Pub. No. 8, Carnegie Ins. Wash., 1907.
’ Whetam, “‘ The Theory of Solution,’’ 1902, Cambridge, p. 170.

TENSION PHENOMENA OF LIVING ELEMENTS. II5

membranes have been found that are impermeable to them.
Overton supposed that the substance, in order to diffuse, must
dissolve in the membrane. Kahlenberg and others consider a
solution as a chemical combination between solute and solvent,
and osmosis as a series of chemical reactions between the mem-
brane and- the two solutions, continuing until equilibrium is
established. The essential points in the theory are: that the
membrane is not a molecule sieve, but a substance with specific
properties, and the chemical characters of the membrane and of
the dissolved substances affect osmosis.

Willard Gibbs found that the more a solute lowers the surface
tension of a solution, the more it tends to pass out of the solution,
1. e., by osmosis, or if this is prevented, to collect at the surface of
the solution. This law has been extensively investigated and
confirmed by I. Traube. For instance, in general, lipoid-soluble
substances lower the surface tension of water and tend to diffuse
out of it, whereas electrolytes slightly raise the surface tension
of water and attract water from the adjacent phase. Osmosis
may occur in opposite directions simultaneously. Gibbs and
Traube state that the greatest osmotic flow is from the solution
of lower surface tension to that of the higher, but this is not
_ generally accepted. Osmosis consists of two distinct processes,
from one solution to the membrane, and from the membrane to
the second solution.

In case the membrane consists of two or. more chemically
different membranes placed one on another, osmosis consists of a
series of steps; and Hamburger! made double membranes through
which certain substances diffuse more rapidly in one direction
than in the other.

Traube calls the bond between solute and solvent the “‘attrac-
tion pressure.’’ In general, attraction pressure of ions increases
with valence. The less the attraction pressure of the solute, the
more it lowers the surface tension and tends to pass out of the
solution. The presence of one solute lowers the attraction
pressure of another in the same solution, and the greater the
attraction pressure of a solute the more it lowers that of another.
We might express this idea by saying that one substance takes

1 Biochem. Zeit., 1908, XI., 443.

116 J. F. MCCLENDON.

part of the solvent away from the second and increases the con-
centration of the second substance. This may explain the effect
of a harmless substance in increasing the toxicity of a-poison.
Schnerlen' observed that a solution of phenol below the threshold
of toxicity for certain bacteria is rendered toxic by adding NaCl.
Stockard showed that the toxicity of pure solutions of salts on fish
eggs is increased by the addition of sugar, although the total
osmotic pressure of the mixture is less than that of the normal
medium.’

Just as Traube’s precipitation membranes are absolutely
impermeable to certain substances, so do living cells show this
selective permeability. For instance, the vacuole fluid or cell
sap of certain plant cells contains colored substances which do
not diffuse into the protoplasm surrounding the vacuoles. If a
cell be placed in a solution of the pigment, the protoplasm
remains colorless. If the protoplasm be squeezed out of the
cell into a solution of the pigment, it does not invariably become
stained. However, if the cell is injured in certain ways, or
dies from any cause, the pigment diffuses out of the vacuoles
into the protoplasm and thence into the surrounding medium.
We might conclude that the protoplasm in general is imperme-
able to the color, but at death it becomes permeable. On the
other hand, Pfeffer® gives evidence for the existence of a mechani-
cal membrane on the surface of the cell and lining the vacuoles.
De Vries‘ placed cells into 10 per cent. KNO; solution colored
with eosin. The plasma membrane and granular plasm died
and stained long before any dye entered the vacuoles. How-
ever, the granular plasm may have absorbed all the dye, thus
preventing its entrance for some time, without the necessity of
any resistance of the vacuole membrane. Since protoplasm may
be squeezed out in the form of droplets and still appears to be
surrounded by membranes, Pfeffer concluded that the membrane
was formed by the contact of the protoplasm with the medium

1 Arch. exp. Path., 1896, XXXVII., 84.

* However the NaCl in Schnerlen’s and sugar in Stockard’s experiment may
have increased the permeability to the toxic substances, as discussed in later
chapters.

’ “ Pflanzenphysiologie.”’

4 Jahrb. wiss. Bot., 1885, XVI., 465.

TENSION PHENOMENA OF-LIVING ELEMENTS. 117

or with cell sap. He supposed these membranes to be the semi-
permeable parts of the cell, and that they became altered at
death. Pfeffer called this membrane on the cell surface the
“plasma membrane.”

Whereas the nuclear membrane and certain vacuole mem-
branes are semipermeable, these are lacking in erythrocytes,
which are therefore good objects for testing the question whether
the protoplasm in general, or merely its surface, is semipermeable.
Hdber! by two very ingenious but complicated methods, one
based on dielectric capacity, determined the electric conduc-
tivity of the interior of the erythrocyte without rupture of the
plasma membrane. Since the conductivity of the interior
(about that of a .2 per cent. NaCl solution) was found to be
many times greater than that of the erythrocyte as a whole, the
membrane must be relatively impermeable to ions. There is
much other, but less direct, evidence that the semipermeability
resides in the plasma membrane, namely: the rapidity of change
in permeability of certain cells, the sudden increase in perme-
ability of a cell after swelling to a certain size (due presumably to
rupture of the plasma membrane), the ease with which mild
mechanical treatment increases the permeability, and the locali-
zation of electric polarization at the cell surface.

Quincke? supposed these membranes to be of a fatty nature.
This idea was carried further by Overton, who considered the
plasma membrane to be composed, not of neutral fats, but of
substances of the class which are called ‘‘lipoids,’’ which included
non-saponifying ether soluble extracts of organs, 7. e., cholesterin,
lecithin, cuorin, and cerebrin. He found* that all basic dyes
were easily absorbed by living cells, but not most of the sulphonic
acid dyes. This corresponded to their solubility in melted
cholesterin, or solutions of lecithin and cholesterin, or particles
of lecithin, protagon or cerebrin. His argument is somewhat
weakened, however, by the fact that cholesterin decomposes
on melting, and that if lecithin is allowed to absorb water its
solvent power changes.

1 Arch. f. d. ges. Physiol., 1910, CXXNIII., 237, and Eighth Internat. Physiol.
Congress, Vienna, I9I0.

2 Sitzber. d. Kon. Preuss. Akad. d. Wissensch. su Berlin, 1888, Bd. XXXIV.

3 Jahrb. wiss. Bot., 1900, XXXIV., 660.

118 J. F. MCCLENDON.

Many of Overton’s critics do not distinguish between lipoids
proper and a host of ether-soluble substances which are also
called lipoids, and of the data which they present we will con-
sider only that on lipoids proper. Ruhland! found that certain
dyes stain plant cells but are not soluble in solutions of cholesterin
(and vice versa). Robertson? observed that methyl green freed
from methyl violet was insoluble in a nearly saturated solution
of lecithin in benzol, whereas it stained living cells. Héber?
obtained Ruhland’s results, when using certain animal cells,
but found that certain nephric tubule cells absorb all dyes that
are not suspension colloids.

Faure-Fremiet, Mayer and Schaeffer* state that pure choles-
terin does not stain with any dyes (contrary to Overton), mala-
chite green (considered lipoid-insoluble by Ruhland and Hdéber)
stains lecithin, and Bismarck brown (considered lipoid-insoluble
by Ruhland) stains lecithin, cholesterin-oleate and cerebrin.
A mere trace. of free fatty acid greatly affects the behavior of
lipoids toward stains.

Mathews’ considers the absorption of dyes by cells as a chemical
process. Since basic. dyes combine with albumin in alkaline
solution, lipoids in the membrane are not necessary for the ab-
sorption of such dyes.

Traube objected to Overton’s hypothesis on the ground that
Overton’s plasmolytic series is the same as found by Brown, who
used the membrane of the barley grain,® and the same as the
series of the attraction pressures of the substances in water.
But Traube admits in his later papers that the chemical character
of the membrane affects osmosis.

We may conclude that, although the plasma membrane of
some cells may be lipoid in character, this has not been proven,
but, in general, it is more permeable the more the diffusing sub-

stance lowers the surface tension of water.

1 Jahrb. wiss. Bot., 1908, XLVI., 1, and Ber. Deutsch. bot. Gesellsch., 1909,
SV. 772.

* Jour. Bio. Chem., 1908, LV., 1.

3 Biochem. Zeit., 1909, XX., 55.

4 Arch. d’Anat. Mic., 1910, XII., 19.

® Jour. Pharmacol. and Exp. Ther., 1910, II., 201.

® But this is not true of all seed coats. Atkins, Sci. Proc. Roy. Dublin Soc., XII.,
n. s., No. 4, p. 35, observed that the membranes of the bean seed are freely perme-
able, semipermeable plasma membranes arising only after germination.

.

TENSION PHENOMENA OF LIVING ELEMENTS. 119

Nathanson! supposed the plasma membrane to be a mosaic
of lipoids and “‘protoplasm,”’ but it is evident that if the lipoid
portion is not continuous, it can not make the cell impermeable
to any substance.

Czapek? states that lipoid solvents cause cytolysis when the
surface tension of the solution is reduced to .68, and concludes
from this that the plasma membrane contains glycerine tri-oleate
since its emulsion reduces the surface tension of water to this
figure. |

The diffusion of water-soluble substances through swollen-
plates, ‘‘gels’”’ or “‘sols’’ of gelatine, varies inversely with the
viscosity (Arrhenius). The great hysteresis of gelatine gels is
taken advantage of to show that diffusion depends on the vis-
cosity and not on the per cent. of gelatine, at the same temper-
ature.®

The absorption of water by a gelatine plate increases its per-
meability, and the temperature and therefore the presence of
substances which affect this swelling of gelatine affect its perme-
ability. Impregnation of colloidal membranes with bile salts,
alcohol, ether, acetone or sugar changes (usually increases)
their permeability. The effects of substances on the rate of
diffusion through gelatine plates, and on their swelling (viscosity)
and melting point are not always quite parallel.

In case the substance added to the membrane is removable,
the change in permeability becomes reversible, which is true
in regard to many of the substances mentioned above. Changes
in non-living membranes are usually only partially reversible or
are irreversible. Denaturalization of a colloid membrane by
heat, heavy metals, or other coagulative agents which induce
chemical changes in the membrane, or the addition of substances
which cannot be removed, produce irreversible changes in
permeability.

That the permeability of the membranes in living tissue is
increased at death is proven by a host of observations. The
electric conductivity increases enormously at death. Contained

1 Jahrb. wiss. Bot., 1903, XXXVIII., 284; 1904, XXXIV., 601, and XL., 403.
2 Ber. deutsch. bot. Gesell., 1910, 28, 480.

3’ Zangger, Asher & Spiro’s Ergeb. der Physiol., 1908, VII., 90.

4 Zangger, loc. cit.

I20 J. F. MCCLENDON.

substances diffuse out, substances in the medium (fixing fluids,
stains, etc.) diffuse in. There is a more general mixing of tissue
substances. Enzymes come in contact with proteids and
autolysis results.

Certain substances are known to increase the permeability
of membranes in tissues of the body. Thus ether, chloroform,
etc., increase the penetration of fixing fluids, and the exit of
contained substances, and the mixing of tissue substances.
In this way they increase autolysis.

II. Osmotic PHENOMENA IN PLANTS.

It is evident that water, salts, carbon dioxide and oxygen
can, at least occasionally, penetrate plant cells, as otherwise
no growth could occur. In case of the higher plants, the same
is true of sugars and other bodies.! Janse? found that so much
KNO; is absorbed by Spirogyra cells in 10 minutes, that it may
be easily detected microchemically with diphenylamin-sulphuric
acid.

Osterhout® grew seeds of Dianthus barbatus in distilled water.
The rate of growth during the several davs of observation was
normal. In nature, calcium oxalate crystals are found in the
root hairs, but are not formed in the distilled water cultures,
showing that the Ca comes from the medium. If placed in
calcium solutions, crystals became large enough to see with the
polarizing microscope in four hours, showing permeability to Ca.‘

Nathanson® found that nitrates and other substances entered
the cell. Ruhland also observed penetration of salts.

Traube-Mengarini and Scala® conclude that salts enter plant
cells only through the partition walls. At these places there
appears an ‘‘acid reaction’’ (bluing of methyl violet). They

1 See Laurent in Livingstone, ‘‘The Réle of Diffusion and Osmotic Pressure in
Plants,’’ 1903, p. 67.

2 Versl. en Medeel. der Konikl. Akad. van afdeel. Naturs., 3. Reeks, IV. part,
1888, p. 333.

3 Zeits. f. phystk. Chem., 1909, LXX., 408.

4 But compare von Mayenberg, Jahrb. f. wiss. Bot., XXXVI., 381, who found
little penetration of salts into fungoushyphe. And see Demoussy, Comptes Rendus,
CXXVII., 970.

5 Jahrb. wiss. Bot., XXXVIII%, 284; XXXIX., 601; XL., 403°

6 Biochem. Zeit., 1909, XVII., 443.

TENSION PHENOMENA OF LIVING ELEMENTS. I2I

interpret this as showing that the anion of the salt unites with
an H ion of an amino group, forming a free acid, and the kation
of the salt unites with the protoplasm. It appears to me that
the basis of this conclusion is very slight.

Permeability may be investigated by a study of plasmolysis,
which consists in the shrinkage of the surface protoplasm away
from the cellulose cell wall, due to the osmotic pressure of the
hypertonic solution of a dissolved substance which does not
penetrate. A regaining of turgor by the cell while in the hyper-
tonic solution indicates slow penetration of the substance. The
plasmolytic method was originated by Nageli, who also noted
that a shrinkage resembling plasmolysis but accompanied by
outward diffusion of dissolved substances, occurs at death or
severe injury to the cell.!

The plant cell is surrounded by an elastic cell wall. The
internal osmotic pressure may be divided into three resultants:
that causing rounding up of the cell is called turgor, that re-
sulting in stretching of the cell wall is sometimes distinguished as
turgescence, and that resisting the surface tension of the cell,
“‘central pressure.”’

The plasmolytic experiments of DeVries? and others’ are
interpreted by them as indicating a selective impermeability
of the plasma membrane to neutral salts.

In the plasmolytic experiments of Overton‘ all salts plas-
molyzed permanently. Non-electrolytes fell in four groups,
thus: Cane sugar, dextrose, manit, glycocoll >urea, glucerin>
ethylene-alcohol, acetamid>methyl-alcohol, acetonitril, ethyl-
alcohol, phenol, aniline, isobutyl-alcohol, isoamyl-alcohol, methyl
acetate, ethyl acetate, butyl aldehyde, acetone, acetaldoxim.
Diffusion of substances of homologous series increased with molec-
ular weight.

Overton ascertained the permeability of plant cells to alkaloids

1“*Pflanzenphysiol. Untersuchungen,’’ 1885.

2 Zeit. physikal. Chem., 1888, II., 415; 1880, III., 103.

3 Cf. Livingstone, ‘‘The Réle of Diffusion and Osmotic Pressure in Plants,”
Chicago, 1903; Janse, Bot. Cenilb., 1887, XXXIL., 21; Duggar, Trans. Acad. Sc.
St. Louis, 1906, XVI., 473.

4 Vierteljahrschrift der Naturforschers. Gesell. in Zurich, XLIV., 88; Jahr. wiss.
Bot., 1900, XXXIV., 660.

I22 J. F. MCCLENDON.

by their precipitation of the tannic acid in the cell sap. Most
alkaloids penetrate rapidly, but only in the form of the free
(undissociated) base produced by hydrolysis. Hence the pene-
tration (precipitation and toxic effect) may be prevented by
adding a little acid to the medium.

Pfeffer had shown that methylene blue is precipitated by tannic
acid in the cell sap of certain plants.

Some discussion has arisen as to whether the mechanism of
the entrance of dyes into plant cells is similar to that of alkaloids.
Overton showed that lipoid soluble basic dyes penetrate easily.
He at first supposed that only the free color base (undissociated)
is able to penetrate the cell.1. Overton found, however, that
triphenylmethane and chinonimid dyes disprove his assumption,
showing that it is at least not general. This question was taken
up again by Harvey? who found that neutral red or methylene
blue, which stain Elodea leaves in tap water, do not do so if just
enough acid be added to the water to prevent any free color
base from forming.

He observed that, although these dyes are not precipitated
in the cell sap of this plant, they become more concentrated in
the cell sap than in the medium. Neutral red is bright red in
the cell sap, indicating that the reaction-is acid (no free color
base is present). He supposes that the absence of any of the
dye in the form of the free color base prevents it from diffusing
out of the cell, hence it becomes more concentrated within than
without.

In using the plasmolytic method, if a cell does not recover
from plasmolysis in a solution of a salt, it is said to be imperme-
able to that salt. However, the cell may recover, but may be
killed by penetration of the salt, and shrink again. It is possible
that Overton and others failed in some cases to note this transient
recovery. Contrary to Overton, Osterhout*? found Spirogyra
permeable to alkali-salts and alkaline earth salts, but more

1 In this connection it is interesting to note that Robertson observed that free
color bases, and to a less extent free color acids, are much more soluble in fats
than are their salts. This is what we should expect, since the salts dissociate in
water, and ions are insoluble in fats.

* Science, 1910, n.s., XXXII., 565.

8 Science, IOLII, Nei: ONO UV 8 7a ROO ET Oe

TENSION PHENOMENA OF LIVING ELEMENTS. 123

easily to Na than to Ca. It is plasmolyzed by .2M CaCl: and
not by the isosmotic .29M NaCl but by .38M NaCl. .r9o5M
CaCl. and .375M NaCl just failed to plasmolyze. On mixing
100 c.c. .375M NaCl with Io c.c. .195M CaCle, thus decreasing
the osmotic pressure of the former, marked plasmolysis occurred.
This indicates that Ca decreases the permeability to Na.!. From
further work by the same author, not yet published, it appears
that Na increases and Ca decreases the permeability of certain
marine plants. Also Fluri? obtained increase in permeability by
salts of aluminium, yttrium and lanthanum.

DeVries plasmolyzed cells of Tvradescantia, containing blue
cell sap, with 4 per cent. KNOs solution, then added nitric acid
until the color changed to red. The acid made the cells per-
meable to KNO; for they regained their turgor and finally burst.
This explains the easy penetration of acids into cells. Pfeffer?
found that if red beet cells, petals of Pulmonaria, stamen hairs
of Tradescantia and other anthocyan-containing cells are placed
in extremely dilute HCl or H.SO,, they suddenly turn red, in-
dicating immediate penetration of the acid. If allowed to re-
main but a short time, the cells are not killed, and the color
change is reversed on returning the tissues to acid-free water.

I have repeated these experiments, using cells of red heet,
red cabbage and red nectar glands of Vicia faba, and find that
mineral acids penetrate, but that (the lipoid soluble) acetic acid
penetrates much more rapidly and also more easily alters the
plasma membrane, causing pigment to diffuse out, if not cau-
tiously applied. Alkalis also penetrate, but (the lipoid soluble)
ammonia penetrates much more rapidly than the others. Am-
monia does not so easily increase the permeability to the pigment
as does acetic acid.

Ruhland?! after staining root hairs of Trianea, etc., with the
indicators, methyl orange and neutral red, found that mineral
acids as well as lipoid soluble acids penetrated.

1 The work of Kearney, Report 71, U. S. Dept. of Agriculture, indicates that
Ca prevents the plasmolytic and toxic effect of Mg, but this is “‘ false plasmolysis”’
following death.

2 Flora, 1908, XCIX., 81.

3 **Osmotische Untersuchungen,”’ Leipzig, 1877, p. 135.

4 Jahrb. wiss. Bot., 1908, XLVI, I.

124 J. F. MCCLENDON.

One defect in the plasmolytic method is the fact that the cel-
lulose cell wall, if not very thick, is elastic, and a slightly hyper-
tonic solution may cause the cell to decrease in volume without
pressing the protoplasm away from the cell wall. This source
of error may be eliminated by substituting calculations of the
volume of the cells (as necessary for animal cells) for observations
on plasmolysis.

It is well known that movement, and in many cases increase
in size of plants is due to changes in turgor of the cells. If we
exclude the turgor changes in aerial plants produced by variations
in the ratio of the water supply to the transpiration, turgor
changes may be due to changes in the osmotic pressure of the
external medium, or of the cell sap (due to metabolic changes)
or to changes in the permeability of the plasma membrane.
Lepeschkin! has confirmed Pfeffer in showing that changes in
permeability of stipule cells accompany (or immediately precede)
changes in turgor. By chemical analysis of the medium he has
shown that an outward diffusion of dissolved substances, from
the cells, accompanies loss of turgor, and by plasmolytic ex-

periments, that the permeability to certain substances increases.

It is interesting to note the force that may be exerted by such
changes in turgor. From measurements of the pull of a stamen
hair of Cynara scolumus or Centaurea jacea on loss of turgor fol-
lowing stimulation, it seems not improbable that the change in
turgor amounts to 2-4 atmospheres (Héber). This also indicates
the strength of the cell wall necessary to prevent rupture of the
plasma membrane. ‘The osmotic pressure of the juices pressed
out of plants varies from 3.5—9 atmospheres.2 The pressing
out of the juices causes an error due to chemical changes; on
the other hand, in taking the freezing point or pieces of plant
tissues, an error arises from lowering of the freezing point by the
walls of the capillary spaces. Miiller-Thurgau* found the A
(corrected freezing point lowering) of plant tissues =.8-3.1°.
Many plants respond to light by definite movements, produced

1 Ber. deutsch. bot. Gesell., XXVI. (a), 725.

2 DeVries, Pringsheime Jahrbucher wiss. Bot., 1884, XIV., 427; Pantanelli, zbd.,

1904, XL., 303.
8 Landwirtschaftl. Jahrb., 1886, XV., 490.

o~

TENSION PHENOMENA OF LIVING ELEMENTS. 125

by turgor changes in certain of their cells. Trondle! found. that
light produced changes in permeability of these cells.

Changes in permeability may not only affect the turgor, but
also the assimilation and excretion, and consequently the
metabolism and growth of the cells. Chapin? observed that
CO, in certain doses is a stimulant to the growth, not only of
green plants but also of moulds. As only a few saprophytes can
decompose COs, it is not probable that its effect is nutritive.
A similar stimulating action of ether and various salts, even
such toxic ones as those cf zinc, was previously known. These
salts probably stimulate without penetrating the cells, since
Zn, for instance, is not a constituent of protoplasm.’ This leads
one to suppose that the initial effect of all of these substances
is on the surface, changing the permeability of the cells.

Wachter? found that potassium decreases the permeability of
onion cells. Sugar diffused out of sections of Allium cepa placed
in distilled water or hypotonic sugar solutions, but a trace of
potassium salt entirely prohibited the diffusion. When the K
was removed the diffusion recommenced.

Czapek® determined increase in permeability by the exosmosis
of tannin in cells of Echeveria leaves. Various monovalent al-
cohols and ketones, ether, ethyl urethan, di and tri acetin,
Na-oleate, oleic acid, lecithin and cholesterin all just caused
exosmosis of tannin in concentrations (aqueous solutions) which
had a surface tension of about 0.68. It would appear therefore
that these substances, chiefly of the class of indifferent narcotics,
alter the cells if they diffuse into them, or diffuse into certain
structures such as the cell lipoids or the plasma membrane.
It seems more reasonable to suppose that the plasma membrane
is the structure affected, and the more the substance lowers the
surface tension of water, the more it diffuses into the plasma
membrane. When this membrane is altered, it allows escape
of tannin. Some substances such as chloral hydrate are effective

1 Jahrb. f. wiss. Bot., 1910, XLVIII., 171.

2 Flora, 1902, XC., 348.

3 Cf. Loeb, ‘‘Dynamics of Living Matter,” pp. 73, 74.

* Jahrb. wiss. Bot., 1905, XLI., 165.

5** Uber eine Methode zur direkten Bestimmung der Oberflachenspannung der
Plasmahaut von Pflanzenzellen,"’ Jena, G. Fischer, tort.

126 J. F. MCCLENDON.

in less concentration, and probably affect the cell chemically
as well as physically.

Mineral acids caused exosmosis of tannin when the concen-
tration just exceeded 1/6,400 normal, and the effect is probably
due to Hions. At this same concentration Kahlenberg and True!
found the growth of seedlings of Lupinus albus to cease. It
appears, therefore, that this cessation of growth is due to in-
creased permeability, causing decreased turgor of the cells.

Changes in permeability may also affect secretion (excretion).
The addition or formation of alcohol or acetates causes yeast and
other fungi to secrete (excrete) for a short time, various sub-
stances, especially enzymes which do not come out in a culture
medium lacking the reagent.2 It appears that the alcohol or
acetates increase the permeability of the fungi to these substances.

My own experiments*® indicate that pure MgCl, solutions
increase the permeability of yeast. A certain per cent. of yeast
and dextrose in .3 molecular MgCl» eliminated CO, more rapidly
than .5M NaCl or .325M CaCle, all which have about the same
freezing points.- Also, the CO: elimination was more rapid in
the magnesium solution than in a solution of the same concen-
tration of MgCl. with either of the other salts in addition, or in a
solution containing NaCl and CaCl, in the same concentrations
as in their respective pure solutions, or in a solution of all three
salts, or in tap or distilled water. In order to determine whether
the magnesium entered the cells I took two equal masses of com-
pressed yeast and agitated one in H,O and the other in a molecular
solution of MgCl, for 5 hours, then washed each rapidly in H2O
by means of the centrifuge. The ash of the magnesium culture
= .048 gram, that of the control = .0466 gram. Evidently
the Mg did not enter the yeast to any great extent, and probably
acted on the surface, increasing the permeability.

Ewart* observed that after placing plant tissue in 2 per cent.
HCl and washing in water its electric conductivity (ionic per-
meability) was increased. If one portion of the plant is stimu-
lated, the stimulus may be transmitted to other portions. In

‘ Kahlenberg and True, Botanical Gazette, 1896, XXII., p. 81.

* Zangger, ‘‘Asher and Spiro’s Ergeb. d. Physiol.,’’ 1908, VII., 144.
® McClendon, Am. Jour. Physiol., 1910, XXVIL., p. 265.

‘“ Protoplasmic Streaming in Plants,” Oxford, 1903, p. 96.

TENSION PHENOMENA OF LIVING ELEMENTS. 127

this way increase in electric conductivity was produced by stimu-
lation of a point outside the path of the current.

Whereas many plants are very sensitive to sudden and extreme
changes in osmotic pressure, Osterhout! found that certain marine
algze thrived when subjected daily to a change from fresh water,
to sea water evaporated down until it crystallized out, and vice
versa. He does not state whether these alge survive extreme
plasmolysis, or whether they are so easily permeable to salts
as not to be plasmolyzed by the saturated sea water or burst
by the fresh water.

For regulation to slight changes in the osmotic pressure of the
medium, a change in size of the cell altering the turgescence, or
tension of the cell wall, is sufficient.

If Tvadescantia cells are placed in a hypotonic solution, they
begin to swell. But soon crystals of calcium oxalate are formed
in the cell sap, and in this way the turgor, due chiefly to oxalic
acid, is reduced.2 It would be interesting to know what is the
source of the Ca. Was it previously in combination with pro-
teids?

The accommodation to a hypertonic medium takes place, ac-
cording to van Rysselberghe, partly through absorption of
substances of the medium and partly through metabolic produc-
tion of osmotic substances, chiefly the transformation of starch
into oxalic acid.’

III. Bio-ELECTRICAL PHENOMENA.

1. In Plants.

Change in permeability of the plasma membrane to ions would
necessarily cause electrical change due to its influence on the
migration of ions. These electrical changes actually occur, and
may be easily studied.

Stimulation or wounding in plants is accompanied by an elec-
tronegative variation of the affected surface. This negative
region spreads in all directions over the surface, but the rate of

1 Univ. of Cal. Pub., Bot., 1906, II., 227.

2 Van Rysselberghe, Mém. d. l’Acad. royale de Belgique, 1899, LVIII., r.
3 Compare von Mayenberg Jahrb. f. wiss. Bot., XXXVI., 381.

128 J. F. MCCLENDON.

propagation! is much slower than the similar process in muscle
or nerve.’

Pfeffer? supposed that the plasma membrane is normally per-
meable to ions of only one sign. Since the normal cell surface
is positive in relation to the cell interior (cut surface) we may
conclude that the plasma membrane is norma’ly more permeable
to kations (less permeable to anions). Just as the negative
variation of wounding is due to the removal or rupture of the |
plasma membrane, so the negative variation of stimulation would,
on the membrane hypothesis, be due to increase in permeability
of the plasma membrane to the confined anions.

An alternative hypothesis is that these electrical changes
result from changes in metabolic activity. The production of an
electrolyte whose anion and kation have very different speeds
of migration (such as an acid or alkali) would cause electrical
changes. But how are we to account for changes in metabolic
activity? There exists varied evidence for changes in perme-
ability, and it is simpler to assume that changes in metabolic
activity and electrical changes are both the result of changes in
permeability.

Kunkel‘ tried to explain the vital electrical phenomena as the
result of the movement of fluids in the vessels of the tissues, but
bio-electrical changes may occur without such movement of
fluids (Burdon-Sanderson).

Kunkel observed in 1882° that the movement of the leaf of
Mimosa pudica is accompanied by an ‘“‘action current,’ or nega-
tive variation of one surface of the pulvinus. Similar results on
Dionea leaves were obtained by Munk® and specially studied
by Burdon-Sanderson.’ It was stated above that Lepeschkin
had shown that the turgor changes in plants were accompanied
or immediately preceded by changes in permeability to certain
substances. The electrical phenomena suggest that the turgor

1 Which is in mimosa 600~1,000 times as fast as the geotropic impulse in a root,

2 Fitting, ‘Asher and Spiro’s Ergeb. d. Physiol.,’’ 1906, V., 155.

3 “* Pflanzenphysiologie.”’

4 Arch. f. d. ges. Physiol., 1881, XXV., 342.

5 See Winterstein’s ‘‘Handbuch der vergleichenden Physiologie,’ III. (2), 2,
p- 214.

6 Arch. f. Anat. u. Physiol., 1876, XXX., 167.

7 Proc. Roy. Soc. London, 1877, XXV., 441; Philos. Trans., 1888, CLXXIX., 417-

TENSION PHENOMENA OF LIVING ELEMENTS. 129

change is accompanied (or immediately preceded) by increase in
permeability of the plasma membrane to anions. Burdon-
Sanderson states that, whereas the movement resulting from
turgor change begins 2.5 seconds after stimulation, the negative
variation reaches its maximum I second after stimulation. This
may be due to the mechanical inertia, or the time required for
the diffusion of substances.

It was stated in the preceding chapter that light changes the
permeability of the plasma membrane, and Waller! found cor-
responding electrical changes due to light, but not always in the
same direction in different plants. This inconstancy in direction
is probably due to the fact that light not only influences the
permeability, but also the assimilation, and changes in assimi-
lation produce electric changes. This is supported by the fact
that Querton* found that assimilation as well as electric change
is most affected by the longer light rays.

2. In Muscle and Nerve?

Ostwald* proposed the hypothesis that the electric phenomena
of muscle, nerve and the electric organs of fish (which may reach
several hundred volts) are produced with the aid of semiper-
meable membranes. The alternative theory of Hermann, which
would account for the current of injury by assuming the pro-
duction of some electrolyte (alkali?) in the wounded region, whose
anions and kations have very different speeds, seems less prob-
ably to be the correct one.

According to the ‘‘membrane theory,’
element is surrounded by a semipermeable membrane allowing
easier passage to kations than to anions. The kations passing
through the membrane are held back by the negative field pro-
duced by the confined anions, but owing to their kinetic energy,
the kations pass out far enough to give the outside of the cell
surface a positive charge. Therefore any portion of the surface
that is made freely permeable to anions becomes electronegative

1 Jour. of Physiol., 1899—'00, XXV., 18.

2** Contribution a l'étude du mode de la production de l’électricité dans etres
vivantes,’’ Travaux de l'Institut Solvay, 1902, V. ,

3Cf. R. Lillie, Amer. Jour. Physiol., 1911, XXVIII., 197.

4 Zeit. physik. Chem., 1890, VI., 71.

’

the muscle or nerve

130 J. F. MCCLENDON.

in relation to the remainder of the surface. This negative
variation may be produced by artificially removing or altering a
portion of the membrane (producing the current of injury) or
as the result of normal stimulation, making it permeable to anions
(action current).

Bernstein resorted to mathematical proof of this hypothesis.
We will not here go into details, but the gist of the matter is
that if the process were as we have imagined it, the electromotive
force of the current of injury, or action current, should be pro-
portional to the absolute temperature. He found this to be
true for temperatures between 0° and 18°, but between 18° and
32° the E.M.F: was found to be too small. The muscle was not
permanently injured by exposure to the higher temperatures
for the length of time necessary for the experiments. Bernstein
explained this discrepancy by the further assumption that at the
higher temperatures the plasma membrane became slightly
more permeable to anions.!

Since the muscle contains a higher per cent. of potassium than
the blood plasma or lymph, it might be supposed that K ions
passed outward through the plasma membrane and gave the
surface of the muscle element the positive charge. But if this
were the case, the current of injury should be reversed by placing
the muscle in a solution containing potassium in greater concen-
tration than in the muscle. This reversal, however, was shown
by Héber not to occur. Since lactic and carbonic acids are pro-
duced by muscle and diffuse out in increased amount on contrac-
tion, one might suppose H ions to give the muscle surface the
positive charge. This is only a guess (and a poor one, since un-
dissociated molecules of COz, and lactic acid are lipoid-soluble)
but may be convenient until some better one is proposed. Per-
haps the carbonic acid combines with amphoteric proteids, which

1 This is similar to the conclusion reached by Biataszewicz, Bull. d. l’Acad. d
Sc. d. Cracovie, Sc. Math. e. Nat., Oct., 1908, p. 783, in regard to the unfertilized
frog’s egg. In order to explain his observation that the rate of swelling in tap
water increased 5 times for every 10° rise in temperature, he assumed that heat
jnereased the permeability to HxO. This would seem to be the simplest explana-
tion, provided the swelling were not due to chemical production of osmotic sub-
stances: and since the A of the ripe ovarian egg is .48° but is reduced to .045° after
oviposition, Biochem. Zeit., 1909, XXII., 390, much if not all of the swelling is
probably due to the initial osmotic pressure of the egg interior.

TENSION PHENOMENA OF LIVING ELEMENTS. I3I

then set free Ht and HCO; ions, thus increasing the ionization
and therefore reducing the number of undissociated molecules,
which can escape.’

Since Osterhout showed that certain electrolytes may alter
the permeability of cells, we might expect to find, on the membrane
hypothesis, an effect of salts on the electric polarization of
muscle. Hdber? observed that a portion of the surface of a
muscle treated with certain salts, KCl for instance, becomes
electro-negative (more permeable to anions) whereas a portion
treated with Nal or LiCl becomes positive (still less permeable
to anions than is the normal unstimulated muscle). The order
of effectiveness of the ions is as follows: Li< Na<Cs<NH;<Rb
<K and CNS<NO;<I<Br<Cl<valerianate, butyrate, pro-
pionate, acetate, formate <SQO,, tartrate. Similar ionic series
were found by Overton, R. Lillie, Schwartz, Mathews, Griitzner,
Hober, and Mayer in the effect of salts on the functional activity
of muscle, nerve and cilia, but the exact relation of these phe-
nomena to permeability is not understood in every case. Pure
solutions of salts of alkali metals may “‘inhibit’’ muscle by in-
creasing permeability, but salts of alkali earth metals are said
to ‘inhibit’? by decreasing permeability... Mayer says that the
effect of salts on cilia is the reverse of that of muscle, but the
relation of this to permeability is not known. Since ions affect
the aggregation state of hydrophile colloids in the same or ex-
actly reversed order, and the kation series is found in no other
known physico-chemical phenomena, it might be supposed that
the semipermeable membranes of muscle are colloidal.

It seems probable that sugar solutions inhibit the activity of
muscle by increasing the permeability, but since sugar is not an
electrolyte this question cannot be tested by electric methods.

A negative variation of muscle may also be produced by the
so-called ‘““hamolytic’’ substances, but is irreversible, whereas
that produced by salts may be reversible. In this connection it

1Roaf, QO. J. Exper. Physiol., 1910, III., 171, supposed the anion to be protein;
however it has not been shown that proteids, or even amino acids diffuse out on
stimulation. I do not see that the speculation of Galeotti, Zeit. f. Allgem. Physiol.,
1907, VI., 99, is at all explanatory.

2 Loc. cit. and Pfliiger’s Arch., 1910, CXXXIV., 311.

132 J. F. MCCLENDON.

is interesting to note that Overton! found the permeability of
muscle to be similar to that of plant cells.

It might appear to the reader that the membrane theory is
merely wild speculation. What proof have we that on injury
or during contraction the muscle is more permeable to any ion?

DuBois Reymond? and Hermann? explained the fact that living
muscle has a greater electric resistance than dead muscle on
the hypothesis that the resistance of living muscle is due to the
presence of membranes, which become more permeable at
death. They demonstrated the resistance of muscle tissue to
the passage of ions by the fact that electric polarization occurs
in muscle tissue on the pasage of an electric current. It seems
to me that Kodis* and Galeotti? take a step backward, in at-
tributing the decreased resistance of dead muscle to the liberation
of ions. Galeotti tried to support his view by determinations
of the freezing points of the living and dead muscle, but found
on the contrary that the change in electric conductivity of the
muscle did not correspond to the change in the osmotic pressure.

Du Bois Reymond® observed that the electric conductivity
of muscle changes on (during?) contraction and Galeotti’ found
it to be greater on strong contraction than on weak contraction,
and least on fatigue-exhaustion or cold-anesthesia. However,
the duration of a contraction is momentary (about 1/5 second for
frog’s muscle) and it is not clear that these investigators measured
the conductivity accurately during such a brief period, in fact
they probably measured it after contraction. Therefore I
decided to repeat these experiments, using a method by which
I could measure the conductivity during the actual contraction
period, as well as in the unstimulated condition.®

1 Pfliiger's Arch., 1902, XCII., 115.

2“ Untersuchungen iiber thierische Electricitat,’

3 Phliiger’s Arch, 13872, Vew223 Ve Suse

4 Am. Jour. Physiol., 1901, V., 267.

5 Zeit. f. Biol., n. £., 1902, XEXV., 289; 1903, XOeVIL- 165.

Soc. Git.

EOC. Git.
8 McClendon, American Journal of Physiology, 1912, XXIX., 302.

’

1849.

TENSION PHENOMENA OF LIVING ELEMENTS. 133

Experimental.

Platinum electrodes, platinized with platinic chloride contain-
ing a little lead acetate, and of a form similar to those designed
by Galeotti, were used. Galeotti stimulated the muscle through
the same electrodes used in measuring the electric conductivity,
by switching on a different electric current. Though it were
possible to throw a switch quickly enough to have the current
for measurement of conductivity pass through the muscle
during contraction, it would be necessary to use a string gal-
vanometer to take the reading, and this method would probably
not be very accurate. A more accurate method is that of Kohl-
rausch, in which a rapidly alternating current reduces polarization
at the electrodes and in the tissue, but it is necessary to throw the
muscle into tetanus in order to have time for the reading. I
accomplished this by using the same current for stimulation and
measurement of conductivity. A very small induction coil was
fitted with a rheostat in the primary. Another rheostat in the
secondary could be thrown out of the circuit by a switch. By
adjusting the rheostats, a current strong enough to be dis-
tinctly heard in the telephone, yet too weak to stimulate the
muscle, was obtained. By switching the resistance out of the
secondary circuit, the current could immediately be increased so
as to throw the muscle into tetanus. Since the Wheatstone
bridge was used, the difference in current strengths had no direct
effect on the readings. The conductivity increased from 6 to
28 per cent. (being usually about 15 per cent.) on stimulation.

We have, then, evidence for the increase in permeability of
muscle to ions during contraction, but what relation has this
to the mechanism of the contractile process? It has been sug-
gested by D’Arsonval, Quincke, Imbert, Bernstein, Galeotti
and others that the increased permeability to ions causes a dis-
appearance of the normal electrical polarization of the elements,
whose surface tension consequently increases, causing them to
round up (shorten). But what are the elements concerned?
It would be confusing to assume them to be the fibers, as then the
function of the complicated internal structure would be unex-
plained. They are probably not the sarcous elements (por-
tions of fiber between 2 Z-lines) as the rounding up of these ele-

134 J. F. MCCLENDON.

ments would elongate the muscle. And even though contraction
were produced by inequality in surface tension, as assumed by
Macallum! the total surface change would be so small as not to
account for the energy liberated in contraction. In order to
avoid this last difficulty Bernstein made use of hypothetical
ellipsoids. These were surrounded by elastic material to account
for elongation of the muscle.”

The great differences of potential (several hundred volts) that
may be produced by the electric organs of fish, is achieved by
the arrangement of the modified muscle plates in series. All of
the plates have the nerve termination on the same side. On
stimulation of the nerve, each plate becomes negative, first on
the nerve termination side, and thus the negative side of one
plate touches the positive side of the next plate. In this way
the direction of the current may be determined by studying the
anatomy of the innervation. This rule, discovered by Pacini,
finds an exception only in Malopterurus, whose electric organ
is supposed by Fritsch to be derived, not from muscle but from
skin glands.

The electric fish are relatively immune to electric currents
passed through the medium. This is not merely an apparent
immunity due to the fish being out of the path of the current, or
the current being short circuited by sea water (in case of marine
fish). I have received severe shocks from a torpedo that was
entirely submerged in sea water.

3. Ameboid Movement.’

The normal unstimulated surface of plant and animal tissues
is electro-positive in relation to the cut or injured surface of
the cells. We have given reasons for assuming that this indicates
greater permeability of the plasma membrane to kations than to
anions, the latter accumulating in the cell interior, gives it a
negative charge.

There are two reasons for believing that this is true also of the
Ameba:

L Science, 0.1S., LOTO Sool S22.

* Meigs., Am. Jour. Physiol., 1910, XXVI., I91, supposes the rounding up of
muscle elements due to increased turgor.

§ McClendon, Arch. f. d. ges. Physiol., 1911, CXL., 271.

TENSION PHENOMENA OF LIVING ELEMENTS. 135

1. If a weak electric current is passed through water in which
an Ameba is suspended, it is carried passively toward the anode,
indicating that it has a negative charge. This charge may be
due to confined anions.

2. If a stronger electric current is passed through an Ameba,
it begins to disintegrate first at that surface nearest the anode.
The disintegration is probably due to the accumulation of ions
retarded by the plasma membrane. The ions in the medium are
free to pass around the Ameba, but the contained ions must pass
the plasma membrane in order to migrate to the electrodes.
Since the disintegration is toward the anode, it is probably due
to anions which cannot get out of the Ameba. Since no corre-
sponding disintegration begins toward the kathode, the plasma
membrane is probably more permeable to kations.

The surface tension of the Ameba is very low, and apparently
increases on strong stimulation (indicated by rounding up of the
Ameba). We saw that stimulation in plant and muscle cells
caused increased permeability to ions, and consequently dis-
appearance of the normal electrical polarization, and thereby
causing increased surface tension. We might conclude therefore
that the low surface tension of the Ameba is caused by electric
polarization, due to the production of some metabolic electrolyte
whose anions cannot escape; and that strong stimulation causes
increased permeability and hence disappearance of the electrical
polarization. :

This would explain all negative tropisms of the Ameba. The
surface tension of the portion most strongly stimulated is in-
creased, and the Ameba flows away from the stimulus.

In order to explain positive tropisms we would have to make
another assumption. If the stimulus did not act directly on the
plasma membrane, but penetrated the Amebva and acted on the
protoplasm, and increased the production of the metabolic
product producing polarization of the plasma membrane, it
would thereby decrease the surface tension. The local decrease
in surface tension would cause the Amebda to flow toward the
source of the stimulus, just as the quicksilver drop in dilute
HNO; flows toward potassium bichromate in Bernstein’s experi-
ment.

136 J. F. MCCLENDON.

All stimuli producing positive tropism would then have to
penetrate to a greater or less distance into the Ameba. But the
same stimulus thus acting on the interior might, in greater
intensity, affect also the plasma membrane, increasing its
permeability and changing the positive to negative tropism.
Such a change of the sign of tropism has been observed.

Soap lowers the surface tension of fats and lipoids, and Quincke,
Biitschli, Loeb, Robertson and others supposed that lowering
of the surface tension of living cells might be due to soap. How-
ever, I found that soap always causes negative tropism in Ameba,
probably because it increases the permeability of the plasma
membrane.

4. The Propagation of the Bio-electric Changes.

On the hypothesis, that the electric phenomena in muscle and
nerve, as well as other animal and also plant tissues, is due to
change in permeability to ions, we might hope to explain the
wave-like propagation of these changes. Since extraneous
electric currents ‘“‘stimulate” all tissues (presumably by in-
creasing permeability) thus causing them to produce additional
electric phenomena, it seems natural that these latter would be
self-propagating. It is probably the negative variation of nerve
which stimulates the muscle, and the negative variation of the
portion of the muscle fiber adjoining the nerve ending, which
stimulates the adjacent portions of the muscle. Nernst found
mathematical proof that electric stimulation is due to change in
ionic concentration at the semipermeable membranes.

I have found evidence that the negative variation (current of
injury) in plants, may strongly affect adjacent cells. If an
electric current of suitable density is passed through plant or
animal tissue, negatively charged colloids in the protoplasm
migrate toward the anode. I have observed this movement in
living cells, and the resulting displaced bodies in histological
sections. In certain cases there may be some doubt whether
the colloids moved toward the anode, or water toward the
kathode, but in others, easily distinguishable bodies such as
chromatin granules or threads moved toward the anode.

If the tip of a root be cut off we observe a negative variation

TENSION PHENOMENA OF LIVING ELEMENTS. 137

of the cut surface. This produces an electric current through
the medium and surrounding tissue. The fact that the current
actually passes through adjacent cells is shown by a displacement
of their contained colloids, identical in appearance with the
displacement produced by the currents used in the above experi-
ments. Némec! apparently observed these changes but did not
correctly describe or interpret them.

The fact that an electric current on increase (make) stimulates
muscle at the kathode, and the fact that the muscle surface is
normally positive in relation to the interior (cut surface), prob-
ably indicates that stimulation is produced by a rapid depolari-
zation of the muscle surface.

If this reasoning be applied to an individual contractile
element, we may assume that the current causes kations to leave
the outer surface of the membrane, and other kations to be
attracted toward the inner side of the membrane, and thus the
polarization disappears or may even be reversed. Just how this
causes an increase in permeability of the membrane is a matter
which we will leave to the future for discussion.

It has been supposed that the stimulated region acts as kathode
to the adjacent portions, and these in turn act as kathodes to
the next portions and so the stimulus is propagated.

Stimulation of a part of the surface, causing it to become more
permeable to ions, depolarizes the adjacent parts of the surface
owing to the fact that confined anions migrate through the
permeable region and neutralize the charges of the kations on
adjacent parts of the impermeable region (see Fig. 1). For this
reason the increase in permeability is propagated.

This explanation of the phenomenon in a single element holds
for a tissue made up of many elements provided these are in
contact, as illustrated by the accompanying Fig. 2. This is
probably the mechanism of propagation of the negative variation
(and ‘‘stimulus’’) in many plant and animal tissues.

This mechanism accounts for the movement of the negative
variation over a surface. But it may be possible for this electric
change to jump from one element, to another not touching it.
The observations on the current of injury, cited above, show that

1“ Reizleitung u .d. reizleitenden Strukturen b. d. Pflanzen,’’ Jena, 19or.

138 J. F. MCCLENDON.

increased permeability of part of a tissue surface, may cause
electric currents to flow through cells some distance from the
wound. ‘These currents probably stimulate the cells through
which they pass, which in turn become permeable and produce
electric currents. This explains the propagation of stimuli

Fie. I.

Anions represented by minus sign, kations represented by plus sign. Arrows
denote the direction of migration of ions. The large circle represents the plasma
membrane, the dotted line denoting the permeable and the continuous line, the
impermeable portion.

through loose tissues, and the structural changes, as observed
by Némec.
The rate of propagation of the ‘‘wound stimulus” is very slow,

sé

whereas that of propagation of the “‘stimulus’”’ (negative vari-
ation) in sensitive plants is more rapid, and that of the nerve
impulse still more rapid. We have not, however, sufficient data
to show whether this is a mathematical objection to the hy-
pothesis.

The streaming movements in plants may be stopped by a
strong stimulus or “‘shock.’’ This stimulus is usually propagated
in one or more directions. Ewart! states that the rate of propa-
gation at 18° in a single elongated cell of Niétella is 1-20 mm.

1 Loc. Ctt:

TENSION PHENOMENA OF LIVING ELEMENTS. 139

per sec., but where it has to pass cell walls .ooI-.03 mm. per. sec.
However, the stoppage of the streaming was his criterion of the
presence of the stimulus, and probably the banking of the stream

+ + + +

ee Sere ee “SRS ie area

Ronn tot

FIG. 2.

The squares represent the plasma membranes of adjacent cells. For further
explanation see Fig. 1.

at one point, soon stopped the whole stream thus simulating
the propagation of the stimulus.

TV. NAaArcosis.

If stimulation consists in increase in permeability, we should
expect anesthetics to prevent this change. The object of this
chapter is to present evidence that may support or refute such a
hypothesis.

Overton observed that warm- and cold-blooded vertebrates,
insects and entomostraca, require practically the same con-

centration of the anesthetic for narcosis. Certain groups of

140 J. F. MCCLENDON.

worms require double, and protozoa and plants six times this
concentration. We might conclude from this that nerves (and
especially medullated nerves?) are more susceptible to narcosis
than are other cells. All groups of worms contain nerves, but
Loeb has shown that certain worms may perform codérdinated
movements after the nerves are cut, hence the higher concen-
tration of the narcotic required to quiet them. However it
should be remembered that over-stimulation causes rounding up
and quiescence of Amwba and muscle may be paralyzed by
increasing the permeability. The growth of plants is increased
by a certain concentration of ether and retarded by a greater
concentration. It may be that true narcosis (decreased perme-
ability) of protozoa and plants cannot be produced by such
substances as ether, etc.

Vertebrate nerve tissues are rich in lipoids (which have similar
solubilities to neutral fats) and it is therefore significant that
Overton and also Meyer! found that the partition coefficient of
anesthetic between olive oil and water corresponds to its anes-
thetic power. Meyer? showed further, that with change of
temperature, the change in the partition coefficient between oil
and water, and the anesthetic power of the substance were
parallel. Pohl, Frantz, Gréhaut, and Archangelsky found that
chloroform, ether, alcohol, chloral-hydrate or acetone, became
more concentrated in the central nervous system than in other
tissues. This is probably due to the absorption of the narcotic
by the lipoids (especially the immense mass of myelin) in the
nerve tissues.

If it could be proven that the plasma membrane consists of
lipoids, this solubility of narcotics might be considered direct
evidence for or against the permeability hypothesis, but lacking
such proof we must first attack the subject from another side.

Héber® observed that ethyl-methane, phenyl-methane, chloral-
hydrate, chloroform and hypnon, in low concentration prevent
the production by salts, of the current of injury on muscle.
He showed that in lethal doses on the contrary these narcotics do

1 Arch. exp. Path. u. Pharm., 1889, XLII., 109.

* Arch. exp. Path. u. Pharm., 1901, XLVI., 338.

® Pfliiger’s Arch., 1907, CXX., 492, 501, 508. Cf. R. Lillie, Am. Jour. Physiol.,
192, XXIX., 373.

TENSION PHENOMENA OF LIVING ELEMENTS. I4I

not prevent but even produce a current of injury, in this way
explaining data which might otherwise seem to contradict the
first statement. Galeotti and Cristina! observed that ether,
ethyl-chlorid, and chloroform produce a current of injury on
frog’s muscle.

We may conclude, then, that anesthetics, in the concentration
producing narcosis, so change the plasma membrane as to
prevent salts from making it permeable to anions. This is
probably also true of nerve, since Héber found that ethyl-
methane in low concentration prevented the sensitizing of
nerve with K»SOx.

Héber has attempted to connect these facts with the lipoid
solubility of narcotics. Moore and Roaf? had observed that
small quantities of such narcotics as chloroform, alcohol, ether,
or benzol, precipitated lipoids extracted from organs and sus-
pended in water. But Héber and Gordon* found that colloidal
solutions of lecithin were not precipitated, but were made trans-
parent by ether or chloroform in high concentration. Similarly,
Goldschmidt and Pribram‘ observed that lecithin suspended in
NaCl solution, which is dissolved by chloral hydrate, methane,
or cocaine, in high concentration, is precipitated by them in low
concentration. On the other hand, Koch and McLean’ state
that chloral, hypnon, acetone, or pure ether, do not change the
size of colloidal particles of lecithin (7. e., make them easier or
more difficult to salt out). Calugareanu® explains the mechan-
ism of the precipitation ot lipoids by anesthetics by the increase
in size of the particles due to absorption of the anesthetic.

Thus there scems to be a parallel difference between the action
of low and high concentrations of anesthetics on muscle and
nerve, and the action of the same on lipoid suspensions, but this
does not hold true for all cases. Moore and Roaf’ conclude that
anesthetics are bound, not only by lipoids, but also by proteids,

1 Arch. allg. Physiol., 1910, X., I.

2 Proc. Roy. Soc. London, 1904, LXXIII., 382; 1906, LN XVIL., 86.

3 Hofmeisters Beitrage, 1904, V., 432.

* Zeit. f. exper. Path. u. Ther., 1909, VI., 1.

5 Jour. Pharm. and Exp. Ther., 1910, I1., 240.

6 Biochem. Zeit., 1910, XXIX., 96.
7 Loc. cit.

142 J. F. MCCLENDON.

and their charactersitic action on the permeability of the living
cell may be due to their action on proteids. In other words, the
plasma membrane may be entirely proteid.

It is well known that during narcosis little or no oxygen is
absorbed by nerve tissue. Verworn and his pupils assumed that
the narcotic directly suppressed oxidation. On the other hand
Mansfeld! supposed that the narcotic dissolving in a lipoid plasma
membrane made it less permeable to oxygen. It would be more
in harmony with the phenomena considered in previous chapters,
to suppose that the narcotic in low concentration decreased the
permeability of the plasma membrane to the anions and molecules
of some acid end product of oxidation, and thus stopped the
combustion. An objection to this hypothesis is made by War-
burg? who found that phenylurethan, which only slightly re-
duces oxidation in certain cells, fertilized eggs, delayed cell
division enormously. With greater concentration of the narcotic,
oxidation was greatly reduced.

V. Osmotic PROPERTIES ‘OF THE BLOOD CORPUSCLES.

Hamburger and Bubonavik* have concluded that the ery-
throcytes are permeable to K, Na, Ca and Mg. However, the
opposite conclusion was reached by previous workers.

Gyrn’s,! Hedin,®> Traube® and others observed that the ery-
throcytes are relatively impermeable to neutral salts (exc. NH,
salts) amino acids, various sugars and hexite, slowly permeable
to erythrite, more permeable to glycerine, and easily permeable
to monovalent alcohols, aldehydes, ketones, esters, ether, and
urea. In general, it may be said that the erythrocyte is perme-
able to lipoid-soluble substances or those that lower the surface
tension of water. Such substances (for instance, ether) become
more concentrated in the corpuscle than in the serum. Saponin
becomes 120, and ammonia 880 times more concentrated in
corpuscle than in serum.’

1 Pfliiger’s Arch., 1909, CX XIX., 60.

2 Zeit. physiol. Chem., LXVI., 305.

3 Arch. internat. de Physiol., 1910, X., 1.

4 P fliiger’s Arch., 1896, LXIII., 86, and Koninkl. Akad. von Wetensch. Amsterdam,
IQIO, p. 347.

5 Pfliiger’s Arch., 1897, LXVIII., 229; 1898, LXX., 525.

6 Biochem. Zeit., 1908, X., 371.

7 Arrhenius, Biochem. Zeit., 1908, XI., 161.

TENSION PHENOMENA OF LIVING ELEMENTS. 143

The erythrocytes are practically impermeable to ions. Stewart?
observed that they offered a great resistance to the electric
current. It is difficult to remove all of the serum from a mass of
erythrocytes, but Bugarsky and Tangl, working independently
of Stewart, obtained sediments of corpuscles having a conduc-
tivity of only 1/50 that of the serum. This indicates that the
corpuscles are practically impermeable to both classes of ions,
for if permeable to ions of one sign, they would probably not be
such good insulators. The electric conductivity of the ash
(made up to equal volume) of the corpuscles is about that of the
serum, although the osmotic pressure of the solution of ash of
the latter is greater.?

Hence an increase in electric conductivity of the corpuscles
(as will be considered below) indicates increased permeability to
ions. After the corpuscle becomes permeable to ions, further
increase in conductivity might be due to liberation of ions from
combinations with colloids in the interior. However many ions,
for instance POu,, cannot be liberated without incineration or other
rigorous treatment. Increase in conductivity of the blood by
laking agents has been proven to be chiefly due to increased per-
meability of the corpuscles, since the conductivity of the serum
never shows so great an increase on the addition of the laking
agent, and is usually diminished (by the hemoglobin) if the cor-
puscles are present.

The portion of the normal corpuscle presenting the greatest
resistance to the electric current is the surface layer, since Héber®
observed that the conductivity of the interior of the corpuscle
(determined by its dielectric value) is many times greater than
that of the corpuscle as a whole. Peskind* caused bubbles of
nitrogen to form within the corpuscle and observed that they were
retained by a superficial membrane. This may be the membrane
which resists the electric current.

The chemical composition of the corpuscle is supposed to bear
some relation to its permeability. Aside from the hemoglobin,
and the rather low water content (60 per cent.) the corpuscle

1 Science, Jan. 22, 1897.

_? Moore and Roaf, Biochem. Jour., III., 155.
$ Pfliiger’s Arch., 1910, CXXXIII., 237.
4 Am. Jour. Physiol., VIII.

144 J. F. MCCLENDON.

is composed of lecithin and cholesterin with a little nucleo-
proteid. It is probable that these lipoids are chemically different
in different species of animals, since Lefmann! observed that the
lipoids of erythrocytes of the same species are not toxic, whereas
those of another species may ke very toxic.

The distribution of these substances in the corpuscle has not
been ascertained. Pascucci? supposed the corpuscle to be a bag
of proteid impregnated with lecithin and cholesterin and filled
with hemoglobin. He found that artificial lecithin-cholesterin
membranes were made more permeable to hemoglobin by the
laking agents, saponin, solanin and tetanus or cobra poison.
Dantwitz and Landsteiner suppose the lecithin to be in com-
bination with protein.

Hoppe-Seyler assumed the hemoglobin to be in combination
with lecithin in the corpuscle, and Bang* has shown that lipoids
may be fixed by hemoglobin. It seems evident that there does
not exist an aqueous solution of hzemoglobin within the corpuscle,
since haemoglobin crystals may be made to form in Necturus
corpuscles without extraction of water. Furthermore, Traube
and Goldenthalt find that haemoglobin has a hemolytic action,
and unless there exists some body within the corpuscle which
antagonizes this action (as serum does) a hemoglobin solution
could not be retained by the corpuscle. Probably all of the so-
called ‘‘stroma’’ constituents, not in combination with the he-
moglobin, form the plasma membrane of the corpuscle.

Under certain conditions, the haemoglobin comes out of the
corpuscles, and the blood is said to be laked. Laking of “‘fixed”’
corpuscles occurs only after the removal of the fixing reagent.
Thus, sublimate-fixed corpuscles may be laked by substances
which combine with mercury, such as potassium iodide, sodium
hyposulphite or even serum proteids. The fact that they may be
laked by heating in water is probably because the nucleo-histone
is not fixed by sublimate. This process is prevented by hypertonic
NaCl solution, presumably on account of its power to precipitate
nucleo-histone (Stewart). Formaldehyde-fixed corpuscles may

' Bettrage chem. Physiol. u. Path., X1., 255.
* Hofmeister’s Beitriége, 1905, VI., 543, 552.
* Ergeb. d. Physiol., 1907, VI., 152.

* Biochem. Zeit., 1908, X., 390.

TENSION PHENOMENA OF LIVING ELEMENTS. 145

be laked by ammoniacal water, at a temperature which must be
higher, the more thoroughly they have been fixed. Ammonia
combines with formaldehyde.

Stewart! supposes that the hemoglobin must be liberated from
some compound before the blood can be laked. We cannot say
that the corpuscle is always permeable to hemoglobin from within
outward. However the corpuscle probably is impermeable to it
from without inward, since it does not take up hemoglobin from
a solution, and after the blood is laked the serum contains hemo-
globin in greater concentration than the “ghosts” do.

At any rate, permeability to hemoglobin appears to be inde-
pendent of permeability to salts, since Rollett? found that laking
by condenser discharges may set free the hemoglobin without the
corpuscle becoming permeable to ions. Stewart® concluded that
the same is true of laking with sodium taurocholate (even after
considering the depressing action of hemoglobin on the con-
ductivity).

Stewart* and others had already shown that blood laked by
minimal applications of such laking agents as freezing and thaw-
ing, heating (to 60°), foreign serum, and autolysis (spontaneous
laking) cause but a slight increase in the permeability to ions,
whereas the continued application of some of these agents, or
especially such violent reagents as distilled water and saponin,
cause a marked increase in electric conductivity. On the other
hand, if saponin is added to defibrinated blood at 0°, the con-
ductivity of the corpuscles to ions begins to increase before any
hemoglobin escapes from the corpuscles.

The liberation of the hemoglobin by some laking agents may
be due to the direct action of the reagent in breaking up the com-
pound in which the blood pigment exists, but is probably some-
times a secondary effect, following increase in permeability to
electrolytes.

It has been shown that many laking agents, lipoid solvents,
saponin unsaturated fatty acids, soaps, and hemolysins (con-
taining lipase) are such as would alter lipoids physically or

1 Jour. Pharm. and Exper. Therapeutics, 1909, I., 49.
2 Pfliiger’s Arch., 1900, LXXXII., 199.

3 Am. Jour. Physiol., X.

4 Jour. Physiol., 1899, XXIV., 211.

146 J. F. MCCLENDON.

chemically, whereas pressure, trituration, shaking, heat, condensor
discharges, freezing and thawing, water, drying and moistening,
salts (including bile salts), acids and alkalis, might act also on
proteids.

Since any treatment which causes great swelling! of the cor-
puscle leads to loss of haemoglobin, it is probable that stretching
or breaking of the surface film increases its permeability. But
laking may occur without swelling, and even crenated corpuscles
may be laked by sodium taurocholate.

Hoéber? observed that the relative action of ions in favoring
hemolysis is: salicylate>benzoate>I>NO3, Br>Cl>SO, and
Kk>Rb>Cs>Na, Li. Since this is the order in which they affect
the aggregation state of colloids, their action is probably on the
aggregation state of the colloids of the corpuscle (proteids or lipoids
or their combinations).

The permeability of formaldehyde-fixed corpuscles to ions, is
greatly increased by extraction of the lipoids with ether, or by
treatment with substances such as saponin, which act on lipoids.
Since the proteids have been thoroughly fixed, it is evident that
they play no part in this process, though they may do so in the
non-fixed corpuscles.

The relation of lipoids outside of the corpuscles to hemolysis
has been extensively investigated, and cannot be fully treated
here. Willstatter found that cholesterin combines with one of
the saponins, destroying its haemolytic power. Iscovesco® con-
cludes that cholesterin combines with ‘soap, and prevents its
toxic action.

Changes in permeability of the corpuscles to ions were studied
chemically before the application of the electrolytic method.
Hamburger? and Limbeck® observed that when COs: is passed
through blood, chlorine passes from serum into corpuscles and
the alkalescence of the serum is increased. On the other hand,
the distribution of sodium and potassium is not changed.®

1 Roaf, O. J. Exper. Physiol., 111., 75, supposes this swelling to be due to ioniza-
tion and hence increased osmotic pressure of hemoglobin.

2 Biochem. Zeit., 1908, X1V., 209, and loc. cit.

5’ Comptes Rendus, Soc. Biol., 1910, LXIX., 566.

4 Zeit. f. Biol, T89L, SomVilLe, AOS.

5 Arch. exp. Path., 1895, XXXV., 309.

§ Giirber, Sitzungsber. physik.-med. Ges. Wurzburg, 1895.

TENSION PHENOMENA OF LIVING ELEMENTS. 147

Koeppe! and Hdéber? explain this process in the following
manner: The lipoid-soluble CO, enters the corpuscle, and by
reacting with alkali albuminates in the protoplasm, gives off
more anions than it does in the serum. During the presence of
COs, the corpuscle is permeable to anions, and the CO; or
HCO;- ions pass back into the serum, being exchanged for Cl-
ions to equalize the electrical potential. Sodium bicarbonate
being more alkalescent than sodium chloride, the titratable
alkalinity of the serum is increased.

This explanation is supported by the following facts: When.
CO: is passed through a suspension of erythrocytes in cane sugar
solution the latter does not become alkaline. If COs is passed
through a mass of centrifuged erythocytes, which are then added
to physiological salt solution, the latter becomes more alkaline
than the serum in Hamburger’s experiment. Any sodium salt
may be substituted for serum, and its anions will pass into the
corpuscles.* Also the number of ionic valences passing into the
corpuscle is constant, 7. e., if sulphate is used only half as many
ions enter the corpuscles as when chloride or nitrate is used.
The process is reversed by removal of the COs.

This same phenomenon has been observed in leucocytes by
van der Schroeff.

There seems to be some relation between hemolysis and ag-
glutination of the corpuscles. Arrhenius! supposed that ag-
glutination by acids is due to the coagulation of the proteids of
the envelope. However, since agglutination is followed by
precipitation, it seems probable that the loss of the negative
electric charge which tends to keep the corpuscle in suspension
and causes it to repel every other corpuscle, is partly responsible
for the phenomena.

The fact that water-laking is preceded by agglutination might
be explained if we assume that increase in permeability to ions
leads to loss of electric charge. The charge may be due to the
charges on the colloids of the corpuscle or to semi-permeability
toions. The corpuscle is very poorly permeable to ions, but may

1 Pfltiger’s Arch., 1897, LXVII., 180.

2 Pfliiger’s Arch., 1904, CII., 196.

3 Hamburger and van Lier, Engelmann’s Arch., 1902, 492.
4 Biochem. Zeit., 1907, VI., 358.

148 J. F. MCCLENDON.

be slightly more permeable to some one ion than to others. If
this ion were more concentrated in the plasma or in the corpuscle,
the lattet would become electrically charged, and a general in-
crease in ionic permeability would lead to a reduction or loss
of this charge. The loss of charge would favor their coming
in contact with one another and their precipitation, but their
cohesion is probably due to some other change, possibly the exit
of adhesive substances, on increase in permeability.

VI. ABSORPTION AND SECRETION.
1. Absorption through the Gut.

If a live vertebrate intestine be filled with one portion of a
physiological NaCl solution, and suspended in another portion
of the same solution, fluid will pass through the wall of the gut
from within outward. Cohnheim! found that holothurian gut
behaves in the same way toward sea water, and the absorption
stops if the gut is injured with chloroform or sodium fluoride.

It might be supposed that the hydrostatic pressure produced
by the contraction of the musculature, is the driving force of
absorption, but on the contrary, Reid? found that the wall of
the rabbit’s intestine behaved in the same way when used as a
diaphragm.

Salt is absorbed by an intestine filled with a very hypotonic
solution of it, and water may be absorbed when the solution is
very hypertonic.

Blood salts enter the intestine when it is injured by an ex-
tremely hypertonic solution, or sodium fluoride, chinin or arsenic.

Grape sugar and sodium iodide may pass from without inwards
through the wall of a normal holothurian intestine.

Traube’ claims that absorption is explained by his observation
that the surface tension of the contents of the gut is less than
that of the blood, but this does not apply to the experiments in
which an identical solution was placed on each surface of the
wall of the gut. Traube* found that the addition of a substance

1 Zeit. physiol. Chem., 1901, XXXIIL., 9.

2 Jour. Physiol., 1901, XXVI., 436.

5 Pfliiger’s Arch., 1904, CV., 559. Cf. Iscovesco, Comptes Rendus, Soc. Biol.,
TOIL, LXXI., 637.

4 Biochem. Zeit., 1910, KXVV., 323.

TENSION PHENOMENA OF LIVING ELEMENTS. 149

lowering the surface tension increased the absorption of ‘NaCl
by the gut.

Absorption is probably due to irreciprocal permeability of the
wall of the gut. Hamburger showed that dead gut and even
artificial membranes showed irreciprocal permeability to certain
‘substances. These artificial membranes were of different com-
position on their opposite surfaces (parchment paper-chrome
albumin, or parchment paper-collodion) and he assumed that
the wall of the gut is composed of two osmotically different layers.
In reality there may be more than two such layers, and the plasma
membranes of the individual cells of the gut may show irreciprocal
permeability.

Traube! showed that the rate of absorption of a substance
by living gut is usually greater the more it lowers the surface
tension of water. The order of ions is: Cl>Br>I>NO;>SO,,
HPO, and K, Na>Ca, Mg. The order of non-electrolytes,
according to Katzenellenbogen? is: glycocoll <urea<acetone,
mann:‘t <erythrite <glycerine <acetamid, methylalcohol, propyl-
alcohol, amylalcohol.

The rate of absorption through dead ox gut according to Hedin
is: Br} >NO;>CI>SO,. and K>Rb>Na>Li>Mg and mannit
<erythrite < glycerine < urethan < glycocoll < amylenhydrate
< glycol < urea < propylalcohol < isobutylalcohol < methyl-
alcohol, ethylalcohol.

The action of poisons on absorption may be due to the alter-
ation of the plasma membranes of the individual cells. Mayer-
hofer and Stein‘ state that even sugar in certain concentrations
increased the permeability of the gut.

2. Osmotic Relation of Aquatic Animals to the Medium.

Fredericq found that the salt content of the body fluids of
marine invertebrates is about the same as that of sea water.
Henri and Lalou® showed that the osmotic exchange between
coelom fluid of sea urchins and holothurians and medium is chiefly

1 Pfliiger’s Arch., CX XXII.

2 Pfliiger’s Arch., CXIV., 522.

3 Pfliiger’s Arch., 1899, LX XVIII., 205.
4 Biochem. Zeit., 1910, XXVII., 376.

5 Winterstein, II. (2), 2.

I50 J. F. MCCLENDON.

water. If the sea water was diluted with 14 vol. of isotonic
cane sugar solution, the salt content of the coelom fluid is very
little lowered in 4 hours, and only traces of sugar appear in it.
The result is the same with isotonic urea (which easily pene-
trates most plasma membranes). But the salt content of the
blood of elasmobranchs and teleosts is about haif that of the sea.

Botazzi and his colleagues observed that the osmotic pressure
of the blood of elasmobranchs is about equal to that of the
medium, the salis in the blood being supplemented by organic
substances, chiefly urea, of which there is 2—3 per cent.

If elasmobranchs are placed in concentrated sea water, the
osmotic pressure of the blood rises, but the ratio of urea to salts
remains the same. G.G. Scott found that changes in the density
and osmotic pressure of the blood of elasmobranchs accompany
changes in the salt content of the medium.

However, in marine teleosts as well as all fresh-water animals
which have been studied in this respect, both salinity and osmotic
pressure of the body fluids are very different from that of the
medium. ;

The osmotic pressure of the blood of marine teleosts is about
half that of the sea, but in fresh-water teleosts it is still less
(but much greater than the fresh water). This indicates that
there must be a change in the osmotic pressure of the blood as
the fish ascends a river. Greene! observed that it took salmon
30-40 days to pass the brackish water, in which time they were
acclimatized to fresh water. After being in fresh water 8-12
weeks, the osmotic pressure of the blood was reduced only 17.6
per cent. This reduction may be partly accounted for by the
absorption of the osmotic substances in the blood by the sexual
glands. In harmony with this view is the fact that the osmotic
pressure of the blood of the female was reduced much more than
that of the male. One salmon, that was very weak and probably
dying, showed 32 per cent. decrease in A of blood. Sumner?
observed that changes in weight and salt content of marine tele-
osts accompany, but are not proportional to changes in the
medium.

U.S. B. F., 1904, XXIV., 445; 1909, XXIX., 1290; Jour. Exp. Zodl., TOL,
IX.
2 Bull. U. S. B. F., 1905, XXV., 53, and Am. Jour. Physiol., 1907, X1X., 61.

TENSION PHENOMENA OF LIVING ELEMENTS. I5I

Overton observed that if the cloaca and mouth of a frog in
fresh water are closed, the frog constantly increases in weight.
This can be prevented by the addition of .7 per cent. NaCl to
the medium. In a hypotonic solution water is constantly ab-
sorbed by the skin and excreted by the kidneys. Fischer’s!
experiment, in which ligature of the leg of a frog caused great
swelling below the ligature is probably to be explained by the
fact that water was absorbed by the skin but could not reach the
kidneys, since the blood circulation was stopped. In regard to
Fischer’s explanation, compare the results of Sidbury and Gies.?
Sumner concluded that in the fish, the gills are the chief seat of
osmotic exchange.

It appears, therefore, that osmosis occurs through the integu-
ment (including gills), kidneys and gut simultaneously, and since
the contents of the gut and kidney tubules are not the same as the
medium, we should not expect an osmotic equilibrium between
the body fluids and the medium. Furthermore, all three of these
membranes may show irreciprocal permeability.

Fresh-water fish and non-migratory marine fish are killed by
great changes in the medium, even though it be very gradual.

Bert maintained that if fresh-water fish are placed in sea water,
the gill capillaries contract and become blocked by the distorted
corpuscles. In naked-skinned fishes, not only the gills are
affected, but water may be lost from the tissues.

Bert and Sumner both agree that the salts in sea water cannot
be replaced by any other substance, without causing the death
of certain marine fishes. Mosso* claimed that when sharks are
placed in fresh water, the gill capillaries become so blocked with
laked corpuscles that physiological salt solution could not be
forced through them. He observed that the differences in the
resistance of certain fish to changes in the salt content of the
medium, corresponded to differences in the resistance of their
blood cells to the hemolytic action of such changes. Sumner,
however, states that this blocking of gill capillaries does not
occur in sharks or marine teleosts in fresh water.

1 Fischer, M. H., ‘‘CG£dema,”’ J. Wiley & Sons, 1910.
2 Soc. Exper. Bio. and Medicine, tort, VII., 104.

3 Biol. Centlb., 1890, X., 570.

4 Proc. Seventh Internat. Zodl. Congress, Boston, 1907.

152 J. F. MCCLENDON.

Sumner showed that as the fish becomes enfeebled by the ab-
normal medium, it becomes more permeable to salts.!. Whether
the direct action of the abnormal medium, or the blocking of
the gill capillaries, produce the increase in permeability, has not
been experimentally tested. However, the gills themselves would
not be asphyxiated by blocking of their capillaries, and it seems
probable that the change in permeability is due to the direct
action of the medium.

We may conclude therefore that the death of the fish results
from the osmotic exchange. This may be sufficient to cause death
while the fish still maintains its normal semi-permeability, or
death may occur only after increase in permeability, due to the ~
direct action of the medium on the osmotic membranes.

A similar increase in permeability may explain Wo. Ostwald’s
observations on fresh-water Gammarus in pure salt solutions.2 He
found that the ratio of the rapidity of death to the concentration
is about constant up to a certain point, above which it is much
greater. This critical concentration has nothing to do with the
osmotic pressure, since it is different for different salts. Perhaps
at this concentration the salt made the membranes more per-
meable.

Schiicking* found that nicotine and strychnine made the skin
of Aplysia more permeable to salts. Since cocain retarded
shrinkage in hypertonic solution, he supposed that the hydro-
static pressure produced by the muscles aided shrinkage. How-
ever the hydrostatic pressure is probably very small, and the
effect might have been due chiefly to an increase in permeability
to salts, produced by the cocain.

3. Secretion of Lymph and Tissue Jutce.

H6éber supposes the raising of the osmotic pressure by the kata-
bolism of the tissues, causes fluid to be drawn out of the blood-
vessels, and states that the lymph in the thoracic duct has a
greater osmotic pressure than the blood.

Traube states that the surface tension of transudates and

1 Cf. Greene, above.

2 P fliiger’s Arch., 1905, CVI1., 568.

8 Arch. Anat. Physiol., Physiol. Abt., 1902, 533.

TENSION PHENOMENA OF LIVING ELEMENTS. 153

exudates is always greater than that of the blood. He cites a
case in which a transudate was caused to be absorbed by injecting
into it a substance which decreased its surface tension.

4. Excretion.

Milk and bile have about the same osmotic pressure as the
blood, but urine is almost dry in some animals; it is usually
hypertonic in man but may be hypotonic.

Traube maintains that the surface tension of the normal urine
is always greater than that of the blood, and that this is the
driving force in excretion.

However, Héber and others suppose that the substances to
be excreted may be formed into solid bodies in the tubule cells,
and thrown out into the lumen.

If lipoid-insoluble dyes are fed to frogs, granules in the cells
of certain segments of the kidney tubule are stained with them.
The dye is not first excreted by the glomeruli and then absorbed
from the lumen by the tubule cells, for if the vena Jacobsoni,
-which supplies the tubules, is ligatured, no staining occurs, al-
though the renal arteries still supply the glomeruli.

The stained granules in the tubule cells are thrown out into
the lumen and pass into the bladder. These granules usually
dissolve to form a slimy substance in the urine, but some of them
may remain intact.

The circulation in mammalian kidneys cannot be controlled
in the same way, but after intravenous injection of a certain
lipoid-insoluble dye, no stain may be detected in the walls of
the glomeruli, although the tubule cells are stained. The stain
in the lumen does not appear above the level of the stained tubule
cells. In the excretion of carmine, it may be found in granules
in the tubule cells and lumen, similar to those found in frog’s
kidneys.

It has been supposed that urea is excreted by collecting in
these granules and passing out with them, but it would be even
simpler to assume that some substance is excreted into the lumen,
which combines with urea and so lowers the concentration of that
in solution, thus accelerating its excretion.

The chief recommendation for the granules is their valve-like

154 J. F. MCCLENDON. -

action, which would account for the secretion of urine against a
concentration gradient, but a simpler mechanism of such a process
is shown in Hamburger’s double membranes.

The blood pressure may aid in the secretion of the water of
the urine, which is eliminated chiefly through the glomeruli,
but its insignificance in the elimination of urea is shown by the
fact that after increasing the volume (and therefore pressure) of
rabbit’s blood 70 per cent. by transfusion, the urea elimination
was not or only very slightly increased.

VII. CELL DIVISION.

Various hypotheses as to the cause of cell division have been
advanced by the morphologists. Hertwig, supposed that when
the ratio of nucleus to cytoplasm is less than normal, the cell
will divide.t Gerassimow? subjected cells of Spirogyra to low
temperatures and other abnormal conditions and obtained an
increased amount of chromatin in some of them. These cells
did not divide until the ratio of nucleus to cytoplasm was as
great as at the time of division of a normal cell.

I found that chromatin is not necessary for cell division.’
After extracting the chromosomes from the starfish egg, I caused
it to divide. In this case the ratio of nucleus to cytoplasm was
zero; however the cell did not continue to divide indefinitely.

There is no easy method of determining the ratio of nucleus
to cytoplasm. Some cells contain large vacuoles whose contents
are not considered as cytoplasm. Eggs contain fat drops and
granules compounded of protein and lipoids. These are not
considered as cytoplasm by all investigators. If the granules and
oil are included as cytoplasm, the ratio of nucleus to cytoplasm
is very small, and yet the egg cell does not divide unless ‘‘stimu-
lated’’ by the sperm or some other means.

R. Lillies observed that chemical substances, which in low
concentration cause the Arbacia egg to divide, in high concen-
tration cause outward diffusion of the red pigment (echinochrome)
and compared this to the laking of erythrocytes.

‘He is not confirmed by Conklin, Jour. Exper. Zodl., 1912, XII., 1.
* Bull. Soc. Imp. Nat., Moskau, 1904, No. I.

* McClendon, Arch. f. Entwicklungsmech., 1908, X XVI, 662.

* BIOL. BULL., 1909, XVII., 188.

_

TENSION PHENOMENA OF LIVING ELEMENTS. 155

This is made more striking by the fact, mentioned first by
Loeb, that hemolytic agents are effective in artificial partheno-
genesis. R. Lillie observed that pure solutions of sodium salts
caused the egg to divide, the order of effectiveness of anions being
C1<Br<ClO3;<NO;<CNS<I. He also found that these salts
could be inhibited by others (CaCls, MgCle), as is characteristic
of the antagonistic effects of salts in physiological phenomena,
and the precipitation of colloids.

I found that the sea urchin’s egg contains fatty substances, and
relatively large amounts of lecithin probably in combination with
proteids. I found that Toxopneustes eggs freed from the jelly-
like coverings, contained about 10 per cent. lecithin (alcohol
extract ppt. with acetone) and about 2 per cent. of an extract
soluble in alcohol or acetone and containing rosettes of fat-like
crystals. . This extract blackened strongly with osmic tetroxide
and effervesced on adding dry Na-carbonate in water, then
emulsified, probably it contained unsaturated fatty acid.

According to a private communication by Mathews, the egg
of the starfish contains lecithin and an unsaturated fatty acid,
but no cholesterin. In this last characteristic it differs markedly
from the erythrocyte. There is no way of determining whether
these substances enter into the composition of the plasma
membrane, but the facts are presented in order to indicate the
possibilities.

We have seen that the exit of hemoglobin is probably not due
to increased permeability to this substance. It is possible that
the same is true of echinochrome. [| found that the echinochrome
in the egg shows a continuous spectrum, whereas that extracted
in various ways shows characteristic bands. It may possibly
be held by chemical combination in the egg.

However I found other evidence for increase in permeability of
the sea urchin’s egg coincident with beginning development:!

1. Fertilized eggs are caused to shrink more quickly than un-
fertilized eggs, with isotonic sugar solution. Presumably the

' fertilized eggs are more permeable to the substances exerting

the internal osmotic pressure.
2. The electric conductivity of the egg increases about 144 when
1 McClendon, Amer. Jour. Physiol., 1910, XXVII., 240.

156 J. F. MCCLENDON.

it is fertilized or made parthenogenetic with acetic acid, indicating
increased permeability to ions.

Lyon and Shackell' and Harvey? observed that methylene
blue and neutral red enter fertilized eggs more quickly than
unfertilized eggs. Harvey supposed that only the free color
base (undissociated) entered, since the addition of a little acid
to the sea water prevented the staining of the eggs.

Mathews’® considered the penetration of stains into the egg
as a chemical process (the stain forming a salt combination with
the lecithin or proteins of the egg surface).

Harvey observed, further, that NaOH penetrates fertilized
more easily than unfertilized eggs, but the eggs are killed by the
alkali.

The fact that the unfertilized frog’s egg continues to swell for
a long time in water (Biataszewitz) whereas the osmotic pressure
of the fertilized frog’s egg is quickly reduced to equal that of the
medium (Backmann and Runnstr6ém) indicates increase in perme-
ability to osmotic substances on fertilization. In this connection
it is interesting to note that Bataillon,t Brachet, and myself®
caused the unfertilized frog’s egg to rotate normally and segment
merely by pricking it.

It has been supposed by various observers that the ‘forma-
tion”’ of the fertilization membrane in very closely related to the
segmentation of the egg. Loeb observed that the sea urchin’s
egg may develop without the formation of a fertilization mem-
brane, and I have confirmed this observation, and shown that
it is very probably wrong to suppose that this is a case of failure
in “‘pushing out”? of the membrane. Apparently ‘‘membrane
formation”’ is not essential for the segmentation of the egg,
although by furnishing protection it may insure the development
of the embryo.

Loeb postulated that an osmotically active colloid exists in
the unfertilized egg, but is so covered with lipoids that it does
not absorb water until it is squeezed out or otherwise exposed

1 Science, 1910, XXXII., 250.

2 Tbid.5) p. 505-

* Jour. Pharmacol. and Exp. Ther., 1910, II., 201.

4 Arch. Zool. Expér., 1910 (5), VI., TOT.

5 McClendon, Amer. Jour. Physiol., 1912, XXIX., 2098.

TENSION PHENOMENA OF LIVING ELEMENTS. 157

at the surface of the egg, at the beginning of development (when
it fills the so-called “perivitelline space’’). I observed that this
substance bears a positive charge (is basic) since it migrates
toward the kathode when an electric current is passed through
sea water containing the fertilized egg.

The unfertilized egg is imbedded in a mass of jelly which is
probably mucin. This jelly bears a negative charge (is acid)
since it combines with color bases.

When the positively charged colloid is exposed at the surface
(on increase in permeability) and comes in contact with the
negatively charged jelly, the two mutually precipitate at their
surface of contact, thus forming the fertilization membrane.
But if all of the jelly is washed off of the egg before the latter is
caused to develop, no fertilization membrane is formed (as I
have observed) because no two oppositely charged colloids are
_brought in contact, but the basic colloid may with difficulty be
seen as a refractive layer, which has been mistaken for a poorly
developed “fertilization membrane.”

The observation of Lyon! makes it appear that catalase comes
out of fertilized more quickly than unfertilized eggs, probably due
to increased permeability.

Lyon observed that CO, came out of fertilized more quickly
than unfertilized eggs, and O. Warburg, Loeb and myself?
observed that oxygen is absorbed more rapidly by the former.
We might ask: Does increased permeability allow increased
oxidation, or is increased oxidation the primary cause of the
increased respiration?

The permeability change is the simplest explanation, and in
what other way could oxidation be increased? Loeb supposed
the sperm carried an oxidase into the egg.’ But no addition of
oxidase is concerned in artificial parthenogenesis, and Loeb
assumed that the oxidase (or other enzyme, kinase?) is held in
the egg periphery and cannot penetrate the egg interior until
the permeability is increased.

In addition to oxygen, oxidase, and escape of COs, hydroxyl

1 Am. Jour. Physiol., 1909, IV., 199.

2 McClendon and Mitchell, Jour. Biol. Chem., 1912, X., 450.

3In this connection it is interesting to note that Masing, Zeit. physiol. Chem.,
1910, LXVI., 265, tailed to find more iron in sperm than in sea water.

158 J. F. MCCLENDON.

ions are necessary for the rapid oxidation of the sea urchin egg
(Loeb), and Harvey showed that the unfertilized egg is practi-
cally impermeable to OH ions of low concentration. The
increased permeability allows hydroxyl ions in the sea water to
penetrate the egg, as shown by Harvey, and, since the sea is
always alkaline, this may explain the increased oxidation.

Asters always develop in the egg before segmentation. In the
normal egg these have some relation to the division of the nucleus,
but even if a nucleus is not present, I have observed that the
cytoplasm constricts along a line on the surface farthest removed
from the centers of the asters.

The constriction of the cytoplasm is probably due to a band of
increased surface tension (or to decreased surface tension at
the poles). This might be caused by local increase in perme-
ability to ions, causing decreased polarization, at the equator
(or increased polarization at the poles, due to increased pro-
duction of the polarizing electrolyte in the asters).

The same reasons that were given for assuming that the surface
of the Ameba is electrically polarized, hold good for the egg.
The first change is probably a general increase in surface tension,
indicated by rounding up of the egg. Later this may become
localized from internal causes and result in cleavage.

Hyde! observed local changes in electric polarization of
Fundulus eggs during cleavage, indicating that surface tension
changes and cleavage are due to this cause.

It has been objected that the segmentation of the egg is not a
typical case of cell division, since the egg cell is ‘wound up”
and ready for some ‘‘stimulus”’ to set it going, whereas tissue
or ‘“‘rest’’ after each division before dividing

”

’

cells must ‘grow’
again.
It may be true that growth is prerequisite to division, but
this cannot be formulated quantitatively. In the spore-forma-
tion of certain organisms, a cell may divide in a relatively short
time into myriads of almost ultra-microscopic cells.

Hertwig may be right, in general, in assuming that the relative
growth of nucleus and cytoplasm influences division, but the
difficulties in proving this have been indicated, and this cannot

1Am. Jour. Physiol., XII., 241. |

TENSION PHENOMENA OF LIVING ELEMENTS. 159

be expressed in chemical terms. It is generally supposed’ that
nucleic acid is a more abundant constituent of the nucleus than
of the cytoplasm, but much evidence has appeared for believing
that it is often present in considerable quantities in the cytoplasm.
Loeb supposed that the segmentation of the sea urchin egg is
accompanied by an “‘autocatalytic’’ synthesis of nucleic acid,
since the nuclei increased in number. But Masing! and more
recently Shackell? by chemical analysis found as much nucleic
acid in the unsegmented egg or I-cell stage as in the blastula
stage.

There is some indirect evidence that increase in permeability
may cause an increased division rate of tissue cells. Though
cell growth may influence division, it is probable that perme-
ability influences growth.

Various “‘stimuli’’ cause increased proliferation of cells of the
germinal layer of the skin. It is commonly known that mechani-
cal stimuli increase growth of the skin.

Bernhard Fisher observed that Sudan III. or Scharlack R*
cause increased proliferation of the epidermis. When the dye
is injected under the skin of a rabbit the skin grows toward the
dye.

Furst* found that gradual increase of temperature caused a
corresponding increase in proliferation of tissue cells (due to
increased chemical reaction and inflammation of the tissue).
But when a certain temperature was reached a sudden jump in
the increase in proliferation was observed without a corresponding
increase in inflammation. This is similar to the phenomenon
seen in unfertilized eggs, where a rise in temperature beyond a
certain point causes segmentation.

It has also been observed that electrical stimulation may
cause increased proliferation of tissue cells.

All of these changes (electrical, thermal, or mechanical stimu-
lation, or treatment with lipoid soluble substances) cause in-

1 Zeit. physiol. Chem., 1910, LX XVII., 161.

2 Science, IQII, n. s.. XXXIV., 573.

3 Which are practically insoluble in water but soluble in fats and lipoids and, as
I have observed, slightly in lipoid-protein combinations.

4See v. Dungern u. Werner, ‘‘Das Wesen Bésartigen Geschwiilste,’’ Leipzig,
1907, p. 65.

160 J. F. MCCLENDON.

creased permeability and segmentation of the sea urchin’s egg.
Therefore, from analogy, we may conclude that increase in
permeability may cause tissue cells to divide.
The ‘‘wound stimulus” to regeneration of tissue may also
cause increased permeability of the cells.
(

‘current of
injury’’ produced by the negative electric potential of a wounded

In a preceding chapter it was shown that the

surface is common to animal and plant tissues. The wounded
cell acts as an electric generator and a current flows through
neighboring cells.

I observed that if a current is passed through living tissue,
which is subsequently fixed and stained, basophile substances
will be found displaced toward the anode. In sections of tissue
adjacent to a wound the extent of the current is indicated by the
displacement of basophile granules. The current affects first
the cells in contact with the wounded cells, then extends in some
directions more than others. Electric currents (‘‘currents of
growth’’) continue for many days after the wound has healed.

Since electric currents cause sea-urchin eggs and tissue cells
to divide and proliferate, probably these bio-electric currents

’

constitute the so-called “‘formative stimulus”’ of regeneration.

Embryonic cells, cells of germinal regions, and cancer cells
are distinguished by their great power of proliferation, or rapid
division. It is probable that the plasma membranes of these
cells are more permeable than those of other tissue cells in the
same medium or under the same conditions.

Cancers have been produced by the action of X-rays (electric
pulsations) on the skin. The cells in the skin were so changed
that they proliferated more rapidly. Similarly, electric changes
have been observed to start the egg cell to rapid proliferation.
There is probably some irreversible change in the permeability
of these cells, which does not, however, make the plasma mem-
brane incapable of subsequent reversible changes in perme-
ability (7. e., the change is unlike what occurs at death of the cell).

The suggestion that cancer cells are more permeable than
tissue cells in general may possibly be of therapeutic importance.
Loeb has shown that fertilized eggs are more sensitive than un-
fertilized eggs to various toxic substances (probably because

TENSION PHENOMENA OF LIVING ELEMENTS. 161

these substances enter the fertilized eggs more easily). “The
same explanation may possibly be applied to the effect of sugar
on certain living cells. The unfertilized eggs of the frog, petro-
myzon, sea urchin and annelid have been caused to segment,
by placing them in sugar solutions. Mayerhofer and Stein! ob-
served that sugar in certain concentrations increased the per-
meability of the gut to certain salts, and in this condition the
gut was more easily injured by the diffusion of substances.

Similarly Stockard observed that sugar increased the toxicity
of pure solutions of salts on the Fundulus egg. Morgan and
Stockard? showed that this was not due to the inversion of sugar
or to the osmotic pressure, and supposed that the sugar might
combine chemically with the salt. It seems probable that the
sugar “creased the permeability to salt. The fact that sugar
in fresh water is toxic whereas the same amount of sugar in
the normal medium (sea water) is not toxic or less toxic, indicates
that the salts within the Fundulus egg are the same as those
outside (in sea water), and increase in permeability to them
does not lead to diffusion while they remain in sea water, but
diffusion takes place in fresh water.’

If it be shown that cancer cells are more permeable, substances
may be found which kill cancer cells more easily than tissue
cells as explained below.

Whereas a certain increase in permeability of the cell seems
to cause division, a very great increase in permeability causes
death (hemolysis, cytolysis, bacteriolysis). It has been shown
that certain lysins are specific for certain cells, probably because
the plasma membranes of these cells differ chemically.

The fertilized egg is more easily cytolyzed than the unfertilized
egg by certain substances. It therefore appears that the more
permeable the cell is in the beginning, the more easily is the
permeability brought to the point which causes cytolysis.

Hence it is probable that certain substances may be found by
which cancer cells can be more easily cytolyzed than normal tissue
cells.

1 Biochem. Zeit., 1910, XXVII., 376.

SEIOLe BULL... TO07, KIIIl., 272.

3In the absence of sugar I have shown that no diffusion takes place in fresh
water. Amer. Jour. Physiol., 1912, XXIX., 295.

162 J. F. MCCLENDON.

It has been shown that narcosis is accompanied by decreased
permeability. On the other hand, certain forms of inhibition
of muscle are accompanied by an increase in permeability.
May certain cells be inhibited in proliferation by an increase
in permeability, too great for cell division but not great enough
for cytolysis? The great oxidation rate in eggs inhibited in
cleavage by very hypertonic solutions as determined by Warburg,
seem to indicate this.

It has been shown that certain tissue cells inhibit the pro-
liferation of others. In the healing of wounds, the epidermis
inhibits the growth of connective tissue. If a wound remains
uncovered by epidermis for a relatively long time, processes of
connective tissue may grow outward, but this is prevented by
the growth or transplantation of epidermis over the wound.

Perhaps the proliferation of the connective tissue is due to
abnormal ‘‘stimuli’’ (bio-electric currents, diffusion of sub-
stances) such as cause proliferation in regenerating tissue gen-
erally. The presence of epidermis over the wound might protect

sé

the connective tissue from these “stimuli.”

The foregoing facts and the speculations based on them may
not be of far-reaching importance in themselves, but they suggest
lines of research, which if followed, it is hoped, will add a great
deal to cell physiology and pathology and be an aid to the under-
standing of many problems in therapeutics.

Reprinted from the American Journal of Physiology.
Vol. XXIX.—January 1, 1912. — No. III.

AN ATTEMPT TOWARD THE PHYSICAL CHEMISTRY OF
THE PRODUCTION OF ONE-EYED MONSTROSITIES.

By J. F. McCLENDON.

[From the Embryological Laboratory of Cornell University’Medical College, New York City
and the U. S. Bureau of Fisheries Laboratory, Woods Hole, Mass.|

ee ENTIAL teratology is one of the oldest branches of the

physiology of development. However, the majority of the experi-
ments have been performed on difficult material, such as the eggs of
frogs, birds, and mammals, and with uncertain results. Stockard !
observed that cyclopia, or the numerical defect in the eyes, may be
easily produced in embryos of the marine fish, Fundulus heteroclitus.
He found the defects to arise, apparently, from the retardation in
the growth of the brain region lying between the Anlagen of the two
optic vesicles. Probably this region is more sensitive than any other
to toxic substances.

No one has recorded cyclopia in Fundulus embryos kept undez
normal conditions. A number of monstrosities develop from the
eggs of fish, especially those, such as the trout, which require a very
high oxygen tension for their development. When the temperature
rises, and consequently the oxygen tension falls, many abnormal
forms appear. However, I know of no observations, except my own,
on the repeated natural occurrence of cyclopia in fish. During two
successive seasons I found about one tenth of one per cent of the
smelt embryos, at Cold Spring Harbor, Long Island, developed in
streams, or in hatching jars of the New York State Fish Hatchery,
to be cyclopic. Stockard observed abnormalities in Fundulus embryos
developed in the fresh water at this place.

I have attempted to find the cause of these abnormalities, but have

1 STocKARD: Journal of experimental zodlogy, 1907, iv, p. 165; 1009, vi,
p. 286; American journal of anatomy, roto, x, p. 360.
289

200 J. F. McClendon.

not completed this work. In fact, only one clue has been found, and
that is the CO» content of the water. The fresh water at Cold Spring
Harbor comes from deep artesian wells and springs, and is heavily
charged with carbonic acid. Dr. R. A. Gortner found more than ten
times as much, CO, in some samples of water that had just emerged
as was found in pond water. Ordinary distillation did not change the
CO, content. It seems probable that the carbonic acid is the cause
of cyclopia in these smelt embryos, but I have found no proof of this
assumption. The eggs are deposited naturally in large masses, with
spaces between the eggs. Those on the surface of the masses develop —
faster, due probably to larger amount of oxygen or light, or smaller
amount of CQs. But in the hatching jars the eggs are separated, ©
and all are exposed to the same conditions.

I produced cyclopia in Fundulus embryos with a number of salts
and with volatile anesthetics. The solutions might be very hyper-
tonic or very hypotonic. On the one hand, distilled water containing
only traces of dissolved substances or substances to which the egg is
freely permeable, were effective, while, on the other hand, 3.4 volumes
of sea water concentrated to one volume were likewise so. It seems,
therefore, that osmotic pressure is not primarily the cause of cyclopia.

However, the eggs decreased in volume when placed in theoreti-
cally isomotic solutions producing cyclopia, tending to show that
these solutions increased the permeability of the egg to the substances
producing the internal osmotic pressure, or turgor. We need only to
suppose that the cells between the eye Anlagen are more easily affected
than other cells of the embryo, to explain the action of the solutions.
The decrease in turgor would retard growth in the same way as a
hypertonic solution prevents development.

It is well known that great increase in permeability means death,
and that death is always accompanied by increased permeability.
In my experiments the concentration of salts or anesthetics causing
cyclopia was always just slightly below the lethal dose (Table 1,
The eggs were placed in the solutions at the two-cell stage, and usu-
ally remained in these solutions until the eyes appeared. Those
apparently cyclopic were removed to sea water and observed more
minutely at a later date, then finally made into histological preparations.

In one case cyclopia was observed in distilled water containing

Chemistry of the Production of One-Eyed Monstresities. 291

minute traces of heavy metals,” and it will be observed from Table I
that hypotonic solutions of NaCl and LiCl produced the same result

TABLE I.
Dist. water solutions. Sea water solutions.
Substance.
Lethal dose. | Cas | Lethal dose. Cyclopic dose.
i ae PORES | ... | 8-9 mol. see
JES Soe 4-.5 mol. .3-.36 mol.| 1.2-1.6 ‘‘ | 1.04-1.36 mol.
oe... 2-25 « ae eee | | ase «
MgCl, - 1 eae 4.5 “ pe A5 * Bee ae
2S OS Ba eae: Se | ee
EIN a = se .006-.01 mol. 006 “ >.01 = ES) | gama
co *.00015-.0002 mol. as | 0025- Tee
Cane sugar ... 1. molecular ae >5 molecular |
Enis a | ee oe be Pere Wb hey) sas
Methylalcohol = .| ..... ere <eeS VOle gee | teres
Ethyl “... .| 3.15-4.75 vol.% | 3 vol. % | 3.5-4.75 vol. % | 2-3.66 vol. %
Lo eg sive | 2 A155,
OS GO es Pee Ape | Se c | 1.6-2.4 “
En er eee es eae n oe tia...

DHEhG: a) oo pi ee | apie | 1/6 saturated | 1/8 saturated

1 At about the same concentration KAHLENBERG and TRUE, Journal of the Ameri-
can Medical Association, July 18, 1896, p. 138, found the growth of certain plants to
cease and showed that this was due to H* ions.

as the traces of metal. The addition to sea water of the chlorides of
Na, Li, or Mg, thus raising the osmotic pressure, produced cyclopia.

2 The traces of metal came from the still, and the experiment was designed
to test the effect of water distilled in metal. The distilled water used in all other
experiments was re-distilled in a fused quartz condensing tube, and there was no
cork, rubber, or other organic material in the apparatus.

292 J. F, McClendon.

Stockard * obtained one cyclops monster by adding MnCl, to sea
water.

It is interesting to note that merely the alteration of the relative con-
centration of the salts already present in sea water produces cyclopia.

In order that these salts should act on the region between the eye
Anlagen after the neural tube has formed, they must first penetrate -
the embryo. According to Overton and others, living cells are rela-
tively impermeable to electrolytes. Therefore I determined the per-
meability of the Fundulus egg under the conditions of the experiments
in which cyclopia was produced. The eggs were impermeable to
Cl ions, but permeable to the kations. By a kationic exchange, the
salt content of the interior could be qualitatively but not quantita-
tively altered (see Appendix IT). ~

Anesthetics belong to the class of substances which freely pene-
trate living cells. They also change the permeability of cells to other
substances. Czapek* observed that many anesthetics increase the
permeability of plant cells to the contained tannin, in ‘‘iso-capillary”’
concentrations. By iso-capillary he designates solutions which have
the same surface tension. The anesthetics which behave in this way
are the so-called indifferent anesthetics, z. e., substances which alter
the cells physically but not, or to a small extent, chemically, and
whose effects are wholly reversible unless the concentration producing
narcosis is far exceeded.

In order to determine whether this rule applied to cyelopia, I made
iso-capillary solutions of various anesthetics and observed their
relative effects. Some difficulties were met in determining the surface
tension of the solutions. According to I. Traube, in homologous
series of anesthetics, a molecule of each member is three times more
powerful in lowering the surface tension than a molecule of the pre-
ceding member. However, Traube’s rule appears to be only approxi-
mate, and difficulties are met in determining the molecular concen-
tration of the solutions. Therefore I measured the surface tension
directly with Traube’s stalagmometer, making corrections for spe-
cific gravity. The apparatus used by Czapek was tried but given up.

Since the surface tension was measured directly, there was no

3 STOCKARD: Journal of experimental zoélogy, 1907, iv, p. 187.
* Czapex: Uber eine Methode zur direkten Bestimmung der Oberflachen-
spannung der Plasmahaut von Pflanzenzellen, Jena, G. Fisher, 1911.

Chemistry of the Production of One-Eyed Monstrosities. 293

necessity of having the substances absolutely pure, although the
character of impurities would be significant. I used absolute’ ethyl
alcohol and pure ether, acetone and phenol, but the other substances
were not pure.

Large glass bottles were partially filled with the solutions, a rela-
tively small number of eggs in the two-cell stage added, and the

TABLE II.

| r
1/6 satur-| 1 per cent
|atedether.| phenol,

}

3.2 per
cent
acetone.

Iso-capillary Sper cent 3.36 per | .2 per cent |

solutions. seek cent ethyl.) amyl.

i, #=| ~ #° |
Undiluted . .| Alldead | Nearly all) Alldead | All dead | All dead | All dead.

Diluted to
three fourths.| “ Z | Monop. -| Cy
Diluted to one

half ae 82 Cy.

ate ee
Diluted to
three tenths .

bottles tightly corked and set aside for forty-eight hours; the eggs
were then transferred to sea water. In the tables, Cy. signifies the
production of true cyclopia (1 median eye), Monop. indicates monop-
thalmia asymmetrica (one lateral eye normal, the other vestigial or
absent).

Table II shows that cyclopia-producing solutions of ethyl and
amyl alcohol, ether® and acetone, are iso-capillary, also the lethal
doses are iso-capillary. Ethyl and amyl alcohol and ether have been
classed as indifferent anesthetics. Acetone may be considered as
almost indifferent chemically, at least to many cells.

All of the solutions of methyl alcohol or phenol that were used
killed the eggs. These substances probably act chemically on the eggs.
Czapek found methyl alcohol to fall into the class of indifferent anzs-
thetics as regards plant cells. It may be objected that the toxic action
of my sample was due to impurities, but evidently a contamination
with amyl alcohol and acetone could not be responsible for the effect,

5 My results with ether are not as exact as with the other substances. The
high vapor tension of ether prevented exact measurements of surface tension of
its solution. If the drops were in contact with circulating air, the concentration
of ether in the surface of the drop was lowered. If the drops fell into an enclosed
space, the ether vapor in the air altered the surface tension of the drop.

294 J. F. McClendon.

since these substances behave as indifferent anesthetics, and their
action is additative.
Feré ® found the teratogenic power of alcohols injected into hen’s

eggs, measured in volumes per cent, to fall in the following series, —

ethyl<prophyl, butyl<amyl. Since, according to Traube, each
member of this series lowers the surface tension of water three times
more powerfully than the one preceding, we may conclude that they
were equally effective in “iso-capillary”’ concentrations. However,
the data of Feré are too inadequate for any final conclusions.

It is interesting to note that Child ’ produced cyclopia in flat-worms,
Planaria, with anesthetics.

APPENDIX I (TABLES).

KCl in sea water:

Molecular concentration . . 3 to .8 9
Result. 2.1 eegatecme eeu 50 per cent All dead.
dead.
Pure NaCl:
Molecular concentration . . 2 3 30 A
GRESUIE 5 cite eee nee eae All hatched. None hatched, Monop.! Cy?
monop.
Molecular concentration . . A 5-1
Result): ic acannon Nearly all _—Alll dead.
dead.
NaCl in sea water:
Molecular concentration . . 9 if, 1.04 1.12
Result: i.5 5 tere geome ears All normal. 59 per cent Cy. Cy.
dead.
Molecular concentration . . nee 1.28 1.36 1.6-2.0
Resulti.:<c. uci, eee 60 per cent Monop. Cy., monop. All dead.
dead, cy.
Pure LiCl:
Molecular concentration . . =) 25 3—.4
Result <7 ueasee erecuael fie are Cy. Nearly all dead. All dead.
LiCl in sea water:
Molecular concentration . . 33 A : 5 6 .66
Result. <.9s) "cae Allnormal. Monop. Monop. Monop. Monop. -
1 Monop. = monopthalmia asymmetrica. 2 Cy. = cyclopia.

® Frere: Cinquantenaire de la Société de Biologie, 1899, p. 360. Reviewed in
BALLENTYNE: Antenatal pathology and hygiene, Edinburgh, Green & Co., 1904,
li, p. 210.

7 Cuitp: Biological bulletin, 1909, xvi, p. 277; 1911, XX, P. 309.

~

Chemistry of the Production of One-Eyed Monstrosities. 295

Pure MgCl:
Molecular concentration . . J A 5 ,
LSU! 3 3c eae 75 percent 95 percent All dead.
dead. dead.
MgCl, in sea water:
Molecular concentration . . 2.8 3 SY ae
ISG 2 53 per cent 57 percent 63 p.c. dead, Cy.
dead. dead, monop. monop, cy.
Molecular concentration . . 34 36 38 A
LL ee Monop, cy. 83 percent 88 percent 97 percent

dead, cy. dead, monop. dead.
CaCl, in sea water:

Molecular concentration . . 2
IRGSTIIE 0 Ss Ga All dead.
Pure CaCl:
Molecular concentration . . 2, 5B
Sea 30 per cent dead. All dead.
Volumes of sea water concentrated to one volume:
1.9 2 2.5 2.65 2.8 2.95 4.
50 percent 42 percent 28percent Cy. 62 per cent Monop. All dead.
dead. dead. dead, monop. dead, monop.,
cy.

APPENDIX II (PERMEABILITY OF THE EGG).

It was thought desirable to know something of the composition,
especially the salt content, of the egg, but the analysis was not com-
pleted. After remaining one hour in sea water, 44.318 gm. of unfertil-
ized eggs were wiped dry with filter paper. After desiccation to con-
stant weight, the dry substance was found to be 20 per cent of the
total. The dry substance contained:

Alcohol-ether extract. ...... 12.2 per cent.
SSO 5 a
Se eHE ee cnn ts sa ee OSS ™

The chlorides in the soluble ash = 4.82 c.c. of a normal solution.
Therefore the chloride content of the water in the eggs (35.576 c.c.)
was about .135 normal, or only .27 as much as in the sea water, which
contained from .49 to .51 normal.

An estimate of the magnesium content may be made from the
following, although the sample was too small for accurate results.

Ten gm. eggs contained 2 gm. dry substance. The ash analyzed for
Mg by the method of W. Gibbs gave .0336 gm. magnesium pyrophos-
phate. From this the magnesium content in the 8 c.c. of water in the

296 J. F. McClendon.

eggs may be calculated as .o3 molecular, or one half as much as in
sea water. |

From these preliminary ash analyses we might infer that the eggs,
or certain regions within them, are impermeable to Cl and Mg ions
and MgCl. However, it appeared to me that this might be deter-
mined by dialysis, especially since these eggs will live in distilled water.

In the following experiments the water was re-distilled in quartz.
The eggs were fertilized and washed in many changes of water for
about two hours. They were then placed in a graduate, and water
or salt solution added to make 100 or 200 c.c., and soaked for a certain
length of time. Three fourths of the total volume was decanted,
evaporated, charred, and analyzed. The eggs were placed in a second
volume of the solution and the process repeated.

Solution. _ |P. CE) Hour. |T. Cl. Cl. Hour.|T. Cl.)

H;0 None
M/4MgSO. ‘None |
H.O0 'None

Nitrates |None
|
|

1 T. Cl. = chlorine by titration.

The above table shows the egg to be impermeable to chlorine ions.
The nitrates were of the same kations as are contained in sea water.

The above table shows a kationic exchange, Na in the medium
being exchanged for Mg in the eggs. No Mg came out of the eggs
in distilled water because the Mg" ions were held back by the electric
charge of the Cl ions in the egg. The ash analysis given above sug-
cests that the egg is normally impermeable to Mg ions, and that the
pure NaCl solution, or Na+K, may increase its permeability to Mg.

In this connection it must be emphasized that it is the plasma
membrane or surface of the protoplasm of the egg that shows this
impermeability, and not the relatively firm egg shell, or chorion. I
have demonstrated the permeability of the egg shell, or chorion, of
fish eggs to several salts by microchemical methods. Loeb * suggests

8 Logg: Biochemische Zeitschrift, 1911, XXXVi, 275.

Chemistry of the Production of One-Eyed Monstrocities. 297

that change in permeability of the egg to salts may be due to the fact
that the micropyle is at times open and at others closed. Since ‘the
micropyle is a hole in the egg shell, its stoppage cannot prevent the
diffusion of salts, as the shell is permeable to them.

Vol. of | ee, Dre |

Solution. || Hour. cip.in || Hour. Mg. precip.| Hour. |Mg. precip.
CBBS- ; milligrams.

H.20 None None

|

N/10 NaCl ‘ FZ ‘Dense cloud Dense cloud

H.0 |
N/10 NaCl
N/10 NaCl

1 Tf we can rely on these small quantities, this represents .005 of the total milli-
grams in the eggs.

APPENDIX III (“Lirarum Empryos”’).

Lithium produces a large per cent of embryos with enlarged seg-
mentation cavities, which were observed and described by Stockard,
who called them ‘‘lithium embryos.” I have confirmed this observa-
tion, and found similar embryos in pure solutions of .38 to .5 molecular
NaCl, and in sea water solutions of ether, acetone, molecular dex-
trose, and .3 mol. CaCle, but in these latter the per cent of the “‘lith-
ium embryos” is less than in solutions of LiCl. These embryos were
studied alive, then prepared histologically and studied as serial sec-
tions. Those produced by lithium did not differ from the others.

Apparently the fluid from the segmentation cavity migrates for-
ward at a later period and comes to lie in front of the embryo, thus
producing a vesicle in the pericardial region. However it is possible
that these two collections of fluid have different origins.

® STOCKARD: Journal of experimental zodlogy, 1906, iii, p. 99.

4
a
‘s
nm
3
:
a
é
;

Reprinted from the American Journal of Physiology.
Vol. XXIX.— January 1, 1912.—WNo. III.

THE INCREASED PERMEABILITY OF STRIATED MUSCLE
TO IONS DURING CONTRACTION.

By J. F. McCLENDON.

[From the Embryological Laboratory of Cornell University Medical College,
New York City.]

CCORDING to the membrane theory of Bernstein,’ the muscle
fibre is surrounded by a plasma membrane or surface film,
allowing easier exit to one or more classes of kations than to the corre-
sponding anions. The kations of some electrolyte which is more con-
centrated within than without come through the surface film and give
the surface a positive electric charge. Destruction or alteration of
this film or membrane causes a negative variation (the affective sur-
face being less positive than the normal surface).

It has been suggested that potassium ions, which are more concen-
trated within the muscle than in the blood plasma, give the muscle
the positive charge. However, one should then expect a reversal of
the electric effects by placing muscle in an isotonic solution of potas-
sium salts. This was shown by Héber? and Overton * not to occur.
R. Lillie * suggests that lactic and carbonic acids are the electrolytes
in question, and the H ions give the muscle surface the positive charge.
The difficulty with this view lies in the fact that muscle, as shown by
Overton, is in general permeable to substances soluble in many oils.
It seems, therefore, that this question is not settled.

If the negative variation or “action current’’ of muscle is due to
increased permeability to any ions, we should expect increased electric
conductivity on contraction. It has long been known that the electric

1 BERNSTEIN: Archiv fiir die gesammte Physiologie, 1902, xcii, p. 521.
? Hoser: Archiv fiir die gesammte Physiologie, 1905, cvi, p. 607.
3 OvERTON: Sitzungsberichte der physikalisch-medizinische Gesellschaft, Wiirz-
burg, 1905, p. 2.
‘ Lituie: This journal, 1909, xxiv, p. 14; 1911, XXviii, p. 197.
302

The Increased Permeability of Striated Muscle to Ions. 303

conductivity of muscle increases at death. Du Bois Reymond ® ex-
plained the electric resistance of living muscle by the presence of mem-
branes, which become altered (permeable) at death. He showed that
muscle becomes polarized on the passage of an electric current. It
seems to me that Kodis® and Galaeotti’ take a step backward in
attributing the increased conductivity of dead muscle to the libera-
tion of ions. Galaeotti tried to support his view by determinations
of the freezing points of living and dead muscle, but found, on the
contrary, that the change in electric conductivity did not correspond
to the change in the osmotic pressure.

I have not found in the literature a clear statement that the elec-
tric conductivity of muscle has ever been measured, actually, during
contraction. The contraction period (about one fifth of a second for
frog’s muscle) is too short for an accurate measurement of conduc-
tivity to be made. Therefore I decided to measure the conductivity
during tetanus.

EXPERIMENTS.

Platinum electrodes similar to those designed by Galaeotti were
used. ‘These were “‘platinized”’ with a solution of platinic chloride
containing a trace of lead acetate. Since the muscle was in contact
with the electrodes, there was danger of rubbing off some of the
platinum black. I found that the black adhered more strongly if the
electrodes were previously roughened by coating with platinum black
and then heating in a flame.

The method of Kohlrausch was used to measure the conductivity.
The smallest sized induction coil made for such experiments was fitted
with a rheostat in the primary, and ofly just enough current was used
as is required to work the interrupter. A second rheostat was inserted
in the secondary, and the current could be reduced until it was not
felt when passed through my tongue.

The muscles of the frog’s thigh were used. They were placed
between, and with the fibres parallel to, the electrodes, and the latter
pressed together until the muscle bulged out on all sides. The elec-

®> Du Bots Reymonp: Untersuchungen iiber tierische Electricitét, 1849.

® Kopis: This journal, 1901, v, p. 267.

’ Garaeortt: Zeitschrift fiir Biologie, 1902, n. F. xxv, p. 289, and 1903, xxvii,
p. 65.

|
|

304 J. F. McClendon.

trodes were then fixed rigidly in position and the preparation placed
in a moist, constant temperature chamber.

The conductivity of the muscle was measured with too little cur-
rent to cause contraction. ‘Then, by cutting out resistance in the
secondary circuit, enough current was passed to throw the muscle
into tetanus and a second reading made. In all cases the conduc-
tivity was greater during contraction.

It might be objected that the heating effect of this current would
change the conductivity, but control experiments on liver tissue and
the tissues of certain plants, in which no change in conductivity
occurred, showed this not to be the case.

Since the muscle at all times entirely filled the space between the
electrodes and extended out on all sides, a change in the form of the
entire muscle would not appreciably alter the conductivity. How-
ever, it is possible that the change in form of the muscle fibres might
slightly alter the conductivity, but it is improbable that it would
account for the large differences observed.

If the change in conductivity were dueto metabolic activity in the
muscle, we would expect different results depending on whether the
muscle was measured first in the stimulated or unstimulated condition,
but no such difference was found. If the change in conductivity is
due to chemical change, the latter must be completely and instan-
taneously reversible.

Experiments were made both in +e spring and the autumn, and a
long series of measurements were made on each muscle. The increase
_in conductivity was greatest in fresh preparations and decreased as
the muscle became fatigued, varying from 28 to 6 per cent.

DISCUSSION OF RESULTS.

The increased conductivity of muscle during contraction may be
interpreted as demonstrating the increase in permeability of some
structures within the muscle to anions (since the muscle appears
already permeable to certain kations). According to the membrane
theory, this causes a reduction of the electrical polarization of these
structures, thus causing increased surface tension and contraction.

In order that the contraction be due to increased surface tension
of any structures, the latter must during relaxation be elongate in

The Increased Permeability of Striated Muscle to Ions. 305

the axis of the muscle. We have such a structure in the anisotropic
segment of an ultimate fibril. It is interesting to note that Dues-
berg ® finds these segments to arise from fat or lipoid-containing
bodies known as chondriosomes, and the presence of fat would account
for the high surface tension between them and the sarcoplasm that
is necessary for contraction.

Bernstein calculated the size of the structures that would be com-
patible with the force of contraction, and concluded that it must be
smaller than any structures seen in histological preparations of muscle.
He therefore postulated hypothetical ellipsoids as the elements in
question.

It is possible that surface tension changes are aided by osmotic
pressure,’ since the movements of plants are due to osmotic changes
following changes in permeability. However, the small size of the
muscle elements makes it impossible to apply botanical methods to it.

8 DurEsBERG: Archiv fiir Zellforschung, 1910, iv, p. 602.
9 Cf. Metcs: This journal, 1910, xxvi, p: 191.

Reprinted from the American Journal of Physiology.
Vol. XXIX. — January 1, 1912.— No. III.

DYNAMICS OF CELL DIVISION. —III. ARTIFICIAL
PARTHENOGENESIS IN VERTEBRATES.

By J. F. McCLENDON.

[From the Embryological- Laboratory of Cornell University Medical College, New York City;
and the Station for Experimental Evolution of the Carnegie Institution of Washington,
at Cold Spring Harbor, Long Island.]

I. By MECHANICAL STIMULATION.

GGS of the wood frog, Rana sylvatica, and the tree frog, Hyla

pickeringii, were caused to segment by momentary pressure.
Cutting the mass of eggs from the uterus to pieces with scissors caused
a few eggs to die and a few others to segment. Compression was a
very unreliable method for causing segmentation, and much more
uniform results were obtained by pricking with a fine needle.’

The females were washed with alcohol followed by a strong stream
of water. The eggs were carefully taken from the uterus so as to
avoid pressure and placed in shallow dishes. In some experiments
the eggs were pricked immediately and then covered with water. In
others the eggs were first covered with water and the jelly allowed
to swell slightly, though the eggs remained adherent to the bottom
and were thus held in position. The glass dish was placed under a
Zeiss binocular microscope and the eggs lightly pricked with the
finest sewing needle.

The extent of the puncture had to be regulated with great care in
order to obtain good results. It seemed necessary merely to touch
the surface of the egg in order to start development, although a punc-
ture that did not result.in a large extra-ovate was permissible. In my
operations on thousands of eggs, some failed to be reached by the

1 BATAILLON: Comptes rendus de la Société de Biologie, ror1, Ixx, p. 562, had
previously caused European frog’s eggs to segment by pricking with platinum
or glass needles. He claims to have developed a few to the metamorphosis. He
interprets Guyer’s results on injecting lymph into frog’s eggs as parthenogenesis
due to pricking, but thinks the serum entering the wound may be essential.

298

Dynamics of Cell Division. 209

needle, whereas some others were pricked too deeply and died. The
jelly offered considerable resistance, and a sudden stab with the needle
seemed less injurious than a slow motion.

Eggs which were pricked just sufficiently, rotated in the normal
manner and segmented. ‘The first cleavage was regular in a small
per cent, and showed varying degrees of irregularity in the remainder.
No regular later cleavages were observed, so that it is impossible to
state how many cytoplasmic cleavages occurred, though divisions of
the nuclei continued for a few days, not, however, at the normal rate.

In some cases the first cleavage furrow passed through the point ~
of puncture, but since it did not do so in all cases, further study would
be necessary to analyze the localization factors.

A control was always kept, care being taken not to subject any of
the eggs to pressure. None of these eggs segmented, but it was noted
in some cases that they finally became wrinkled and apparently
decreased in volume. According to Biataszewicz,? the unfertilized
frog’s egg swells continuously. The water which I used came from
an artesian well, and contained over ten times as much COs, as was
found in pond water,’ and it is possible that the CO, injured the eggs.
In harmony with this view is the fact that there was a large mortality
among fertilized eggs developing in it.

II. By ELectricaAt STIMULATION.

Eggs of the wood frog, Rana sylvatica, the tree frogs Hyla picker-
ingii and Hyla versicolor, and the toad, Bufo lentiginosus, were caused
to segment by electric stimulation. The character of segmentation
was similar to that in pricked eggs. A high per cent could be caused
to segment. The segmentation rate seemed to be about normal, for
instance the first cleavage in Hyla pickeringii occurred from two to
three hours after electric stimulation, at 21°C. But in order to avoid
the slightest possibility of contamination with sperm, no males or
fertilized eggs were allowed in the laboratory during the experiments,
and the rate of development was not directly compared with the
normal.

* BIATASZEWICz: Bulletin de 1’Académie des Sciences de Cracovie, 1908,
Pp. 783.
* From analysis by R. A. GoRTNER.

300 J. F. McClendon.

The females were washed with alcohol followed by a strong stream
of water. The eggs were carefully taken from the uterus, without
pressure, placed in glass dishes and covered with water. An alter-
nating current of 110 volts and 60 cycles was passed through the
water from platinum or carbon electrodes held from 2.5 to 15 cm.
apart. The use of an alternating current prevented appreciable
polarization and chemical changes at the electrodes, but in order to
avoid the possibility of the slightest chemical influence, the water
was poured off of the eggs immediately after removal of the electrodes
and fresh water added. Eggs very close to the electrodes were neces-
sarily more affected by the current than those farther removed, but
in comparing experiments I have regarded only those eggs midway
between the electrodes. The position of the electrodes was marked,
and the eggs remained adhering to the glass in their original positions.

An instantaneous exposure to the current was sufficient and pro-
longed exposure injurious, although the resistance of the eggs to
injury varied with the species. Having the electrodes 5 cm. apart,
the injurious effect was seen in Hyla pickeringii eggs after five seconds,
but in toad’s eggs only after ary to one hundred and twenty
seconds’ exposure.

The majority of the experiments were performed in artesian water.
The fact that the use of water re-distilled from barium hydrate gave
as good results, indicates that the CO, of the artesian water was not
essential to parthenogenesis. The addition of 1/50, 1/20, 1/10, or
even 1/5 vols. of sea water did not prevent cleavage. Also the fol-
lowing pure salts and combinations were permissible: NaCl, CaCl,
MgCl., NazCO;, CaOH, chlorides of Na & Ca, Ca & Mg, Na & Ca
& Mg.

A large number of eggs of each species were removed at various
periods after stimulation and prepared histologically in serial sections.
The following account of the internal changes in eggs of the wood
frog applies to all except for details as to size, amount of pigment, etc.

The female pro-nucleus, in passing from the surface at the animal
pole, toward (but not reaching) the centre of the egg, leaves a pig-
ment track similar to that left by the sperm nucleus in fertilized eggs.*
I have no doubt that this pigment track is present in normal develop-

‘In Hyla pickeringii, where there is little pigment, the track is hardly
noticeable.

Dynamics of Cell Division. 301

ment, but has not been observed because the first cleavage furrow
cuts through it longitudinally, as it is being formed, and obliterates
it. In parthenogenetic eggs the furrow is usually not through the
axis of symmetry, in which the pigment track lies, and therefore
usually does not obliterate it.

Although the first cleavage is not usually through the axis of sym-
metry, it is often near this axis, and therefore very nearly regular.
Sometimes a large cell is cut off from a small one. In the majority
of cases, however, two and sometimes more furrows appear simul-
taneously. This is not due to a corresponding increase in number of
nuclei, as sections show but one nucleus, which is the undivided
female pro-nucleus. The metaphase of the first nuclear division is
reached long after the first cleavage furrow begins to cut through
the egg.

The first cleavage usually does not completely cut through the
vegetative pole. Later cleavage furrows sometimes appear, but are
likewise never completed.

Cleavage of the cytoplasm is brought to a standstill, and the nuclear
changes continue. The nucleus divides repeatedly, filling the egg
with daughter nuclei. Finally the egg becomes vacuolated and is
dead within a few days.

In a preliminary note® I have mentioned certain oiabeabineies 1 in
regard to the theoretical significance of these results. However, since
more data are desirable, I will not continue the discussion here, but
hope to do so at a later date.

5 McCLENDON: Science, 1911, N. S. xxxiil, p. 629.

Reprinted from THe JouRNAL OF BroLocicat Cuemistry, Vol. X, No. 6, 1912

HOW DO ISOTONIC SODIUM CHLORIDE SOLUTION AND
OTHER PARTHENOGENIC AGENTS INCREASE
OXIDATION IN THE SEA URCHIN’S EGG?

By J. F. MCCLENDON anp P. H. MITCHELL.

(From the Embryological Laboratory of Cornell University Medical College,
New York City, the Physiological Laboratory of Brown University, Provi-
dence, R. I., and the U. S. Bureau of Fisheries, Woods Hole, Mass.)

(Received for publication, November 4, 1911.)

Loeb has shown that —OH ions favor development.! Our own
experiments (Table 6) as well as those of O. Warburg? demonstrate
that increase in the alkalinity of the medium increases oxidation
in fertilized eggs.

According to Warburg, the egg is impermeable to ~OH ions or
fixed alkalies, because, although eggs stained with neutral red
are changed to yellow by NH; of a concentration which increases
the oxidation only one-tenth, similar eggs are not changed to
yellow by fixed alkalies of a cancentration at which oxidation is
greatly increased.

These facts are, however, capable of another interpretation.
The egg is filled with lipoid particles which take up the neutral
red to such an extent as to render the surrounding protoplasm and
sea water colorless. The —OH ions cannot freely enter the lipoids
in order to change the color of the neutral red. On the other hand
ammonia is lipoid-soluble and can enter.

It might be objected that the ammonia cannot react with the
dye in the non-aqueous medium owing to the suppression of ioni-
zation, but whether the dye is driven out of the lipoids or changed
to yellow in situ, the fact remains that it does become yellow.

Harvey? observed that the addition of but a small quantity of
alkali to sea water containing fertilized eggs stained with neutral

1 Loeb: Chemische Entwicklungserregung des tierischen Eies. Berlin, 1909.
* Warburg: Zeitschr. f. physiol. Chem., lx, p. 305, 1910.
3’ Harvey: Journ. of Exp. Zoology, x, p. 507, 1911.

459

460 Oxidation in the Sea Urchin’s Egg

red, changed the eggs to yellow. However, the eggs were injured
by the alkali and probably their permeability was increased. It
is possible that alkalies or OH ions in any concentration enter
the fertilized eggs but must be present in sufficient concentration
to set free ammonia or change the lipoids in order to affect the dye.

We may assume, then, unless more conclusive evidence indicates
the contrary, that the ~OH ions increase oxidation after penetrat-
ing the egg.

One of us has shown that unfertilized sea urchin’s eggs are poorly
permeable to salts and their ions, but become more permeable
after fertilization or the initiation of parthenogenetic develop-
ment.!. Not only is the permeability increased but the oxidation
rate is increased. Warburg observed that oxidation increases
from five to seven times on fertilization (compare our Table 5,
experiment III). He found also a large increase after the initia-
tion of parthenogenetic development caused by hypertonic sea
water,? fatty acid, alkalies or traces of the heavy metals,* silver
or copper. Our own experiments, given below, confirm and extend
these findings. There appears then to be some relation between
permeability and oxidation, and the present paper is an attempt
to determine what this relation is.

The living cell may be compared to a furnace, and R. Lillie+
advanced the view that increase in permeability opens the draughts
so to speak, allowing the escape of carbonic acid, and hence oxi-
dation is increased. He supposes that the accumulation of car-
bonie acid and perhaps other end-products checks the oxidation,
and increase in permeability to carbonic acid allows oxidation to
proceed. The difficulty with this hypothesis lies in the fact that
living cells have been shown by Overton and others to be freely
permeable to substances which are easily soluble in fats and oils,
or especially in lecithin and cholesterin. Carbon dioxide-is solu-
ble in oils and probably enters cells easily, at least there is evidence
to show that red blood corpuscles are freely permeable to this
gas. Fatty oils are permeable only to the undissociated mole-
cules and not to the ions. Since the proportion of ions of carbonic

‘ McClendon: Amer. Journ. of Physiol., xxvii, p. 240, 1910.
* Warburg: Zeitschr. f. physiol. Chem., \xii, p. 1, 1908.

’ Warburg: Jbid., xvi, p. 305, 1910.

‘ Lillie: Biol. Bull., xvii, p. 188, 1909.

VOLUME (DILUTION)

J. F. McClendon and P. H. Mitchell ABI

acid would ordinarily be small, what conditions in the egg might
favor ionization of CO.?

Not all of the alkali metals in the egg are combined with min-
eral acids; some are combined with proteins. This has been shown
by the senior author with electric conductivity measurements of
hens egg yolk given in the accompanying curves. The continu-
ous line represents the conductivity of yolk freed from protein
granules, and the dotted line, yolk containing an excess of protein

-001 -002 -003

ELECTRIC CONDUCTIVITY

granules, separated by the centrifuge. The granules impede the
current as shown by the fact that the granule-containing yolk is a
poorer conductor than granule-free yolk. But on dilution, the
granule-containing yolk becomes the better conductor. There-
fore ions are set free from the granules on dilution.

The carbonic acid formed within the egg would react with
the alkali albuminates with the formation of alkali carbonates
and bicarbonates, which, notwithstanding hydrolysis, would
liberate a considerable quantity of carbonic acid anions.’ The
dissociated carbonic acid, being unable to escape from the unfer-
tilized egg would lower the —OH ion concentration and thus reduce
oxidation. As the undissociated molecules of carbonic acid escape,
more are formed by the slow oxidation in the egg.

On fertilization, the permeability to the anions of carbonic
acid is greatly increased. They migrate out of the egg, and nega-
tive ions enter the egg to take their place. Since the ~OH ions of
the sea water are the fastest negative ions, they enter the egg and

1 At about molecular concentration the equivalent electric conductivity
of NaCl is 76; of NasCOs, 45, at 18°.

462 Oxidation in the Sea Urchin’s Egg

increase oxidation. However, if some ~Cl or ~SO, ions entered
the egg they would tend to decrease the —OH ion concentration
within the egg. But the senior author has shown that the ferti-
lized Fundulus egg is impermeable to ~Cl ions, for when placed in
distilled water, or in solutions of nitrates or sulphates, practically
no chlorine comes out of the eggs. We may assume, therefore,
that carbonic acid accumulates in the unfertilized egg until the
reaction is neutral or slightly acid. But on fertilization, the per-
meability to carbonic acid anions is increased and the concentra-
tion of this acid is diminished so that the reaction is neutral or
slightly alkaline. The increased alkalinity is the cause of the
increased oxidation. We will now see to what extent our experi-
ments bear out this assumption.

We observed that oxidation is about doubled when the egg is
made parthenogenetic with carbonated sea water (Table 5, experi-
ment II) or alkaline isotonic sodium chloride (Table 3, experi-
ments I and II). In some eases the eggs, being physiologically
different from those in other experiments, did not show the mor-
phological signs of development in this solution, and oxidation was
not doubled (Table 2: Table 3, experiment III; cf. Table 5, experi-
ment I). The eggs begin development while in the alkaline solu-
tion, but eggs made parthenogenetic by treatment with neutral or
acid solutions (neutral sodium chloride or carbonated sea water)
begin development only when transferred to natural sea water or
other alkaline solution. This bears out our hypothesis, for if the
increased permeability remains after the egg is returned to an alka-
line medium, a chance is given for an increase in alkalinity in the
interior, which was lacking in the non-alkaline solution.

Certain facts may seem to contradict our assumption, but prob-
ably they merely limit its application:

1. A slight increase in —OH ions may cause even the unfertilized
egg to absorb more oxygen (Table 2) and a greater increase causes
it to develop. This does not necessarily show permeability of the
unfertilized egg to hydroxyl ions. The increased alkalinity
slowly causes an increased permeability of the egg and thus leads to
parthenogenesis, but the degree of alkalinity of the medium neces-
sary to induce development of the unfertilized egg is far greater

‘These experiments will be published later in the American Journal of
Physiology. :

J. F. McClendon and P. H. Mitchell 463

than that necessary for the development of the fertilized egg or
the egg already made parthenogenetic.

2. The fertilized egg of Arbacia punctulata (but not of some other
sea urchins) may develop in a natural medium, as Loeb observed
and which we have confirmed. In other words, a hydroxyl ion
concentration in the medium, greater than that of distilled water,
is not necessary for development of this egg made freely per-
meable to carbonic acid. However this fact does not set a limit
to the alkalinity of the egg interior. The egg probably contains
more Na than Cl ions, and if it be impermeable to Na or Cl, the
escape of carbonic acid might cause the egg interior to become
alkaline or at least neutral. Eggs made parthenogenetic in some
ways (neutral sodium chloride, for instance) do not develop unless
transferred toan alkaline medium, but this may be due to the possi-
bility that these parthenogenetic eggs are not quite as permeable
as are fertilized eggs. The same may be inferred from oxidation
measurements. Neutral sodium chloride causes the unfertilized
egg to absorb more oxygen than it does in sea water (Table 4)
but the increase is slight, and morphological development does not
commence. If the eggs are then transferred to sea water or other
alkaline solution, some of them may develop.

It appears therefore that increase in permeability is a gradual
process. Although some eggs are so permeable as to be able to
develop in an neutral medium others are less permeable and do
not develop, or develop only in an alkaline medium. By treating
eggs with parthenogenetic agents in various concentrations or for
various lengths of time we may induce various degrees of per-
meability. Even fertilized eggs may be made more permeable by
treatment with parthenogenic agents, and a corresponding increase
in oxidation may be observed (Table 6). In these experiments
the oxidation of the eggs in sea water was measured about ninety
minutes after fertilization: they were then placed in isotonic,
alkaline sodium chloride solution, in which the oxidation increased
one-half, when returned to sea water the oxidation fell below its
previous level in the same medium. According to Loeb, this
indicates death of some of the eggs (20 per cent).

The experiments just described explain the discrepancy between
the results of Warburg and those of Loeb. Warburg! found that

1 Warburg: loc. cit.

464 Oxidation in the Sea Urchin’s Egg

the oxidation of the fertilized egg in isotonic sodium chloride solu-
tions containing a trace of sodium cyanide, is much greater than
in sea water containing the same concentration of sodium cyanide.
Loeb! confirmed this determination, but observed further that
if the cyanide is omitted (from both) no increased oxidation in
sodium chloride solution occurs. The cyanogen in both sea water
and sodium chloride solution depresses oxidation. Since sodium
cyanide liberates "OH ions, we may conclude that the increase
in oxidation in the sodium chloride solution used by Warburg was
due to the increased penetration of hydroxyl ions, following in-
crease of permeability.

In our experiments no cyanide was used, and the alkalinity of
the sodium chloride solution was not greater than sea water, yet
oxidation was increased. In Loeb’s experiment the tendency of
increased permeability to increase oxidation was counteracted
by the effect of lower alkalinity, which decreases oxidations.

Alkaline sodium chloride solution also favors oxidation in eggs
that have reached later stages of development, morula or blastula
(Table 7). In this experiment, the rate of oxidation in sea water
was rising gradually (see next section) before the eggs were placed
in the alkaline soduim chloride solution, but in the latter a sudden
increase of more than 50 per cent was observed.

MATERIALS AND METHODS.

The eggs of the sea urchins, Arbacia punctulata, were used. The
animals were washed in a strong stream of fresh water and opened
with precautions against introducing spermatozoa among the
eggs. The ovaries were removed and placed in the first solution
to be used, sea water or neutral van’t Hoff’s solution. The mass
was strained through bolting cloth of such a grade as to allow but
one egg to pass through one mesh at a time. The eggs were
repeatedly precipitated by gravity in fresh portions of the solution
in order to remove coelomic fluid cells (elaeocytes), and trans-
ferred with a small quantity of fluid to the determination flask.

The rate of oxidation in the various solutions was measured by
comparison of the dissolved oxygen in the solution before and
after the eggs had been suspended in it during a definite period.

1 Loeb and Wasteneys: Biochem. Zeitschr., xxviii, p. 340, 1910.

J. F. McClendon and P. H. Mitchell 465

Winkler’s thiosulphate method of oxygen determination (iodo-
metric), as described in Treadwell’s Quantitative Analysis, was
used.

From 3 to 7 cc. of eggs were used in each experiment, but the
actual volume of the eggs was not measured until after the oxygen
determinations.

We tried a number of methods for filling the determination flask
and sample bottles without an uncertain loss or gain of oxygen.
Loeb collected the water in the sample bottle under petroleum.
Although petroleum absorbs five times as much oxygen as water
does, the oil would tend to reduce currents adjacent to the air-
water surface, and thus reduce oxygen exchange. Using paraffin
oil, we found that it was extremely difficult to prevent a little oil
from sticking within the flask, and abandoned the method. Per-
haps kerosene would have worked better, yet the quantity of kero-
sene that would dissolve in the water might vitiate the experiments.

Since the sea water and the solutions were shaken up and satu-
rated with air at the given temperature before beginning the exper-
iment, the control sample might have been taken under air, with-
out change. But after loss of oxygen in the determination flask,
a gain in oxygen would result from such treatment. By intro-
ducing the solution through a tube passing through a doubly
perforated stopper, and extending to the bottom of the sample
bottle, the exposed surface of the water was made as small and
quiet as possible. By maintaining a constant rate of flow the
error could be made to bear an approximately constant ratio to the
oxidation, no matter whether the sample bottle was filled with air
or some other gas. We tried the effect of introducing the sample
under air, and also under hydrogen, and decided that the latter
method was preferable for oxygen-low samples. In order to make
all errors fall in the same direction we alse collected the oxygen-
high samples under hydrogen.

The experiments were so regulated that the oxygen content of
the determination flask at the end of the exposure would not fall
very low. However, Warburg failed to observe a decrease in the
rate of oxidation in low oxygen concentration.

The water was forced out of the determination flask rapidly into
the sample bottle by hydrogen under pressure. The determination

466 Oxidation in the Sea Urchin’s Egg

flask held 332 ec., the two sample bottles 152 and 142.6 cc. respec-
tively.

The determination flask was placed in a thermostat which was
kept 2° above the temperature which the air had reached at the
beginning of the experiment. In most cases the time of exposure
was one hour. During the first half-hour the eggs were distributed
throughout the solution once every five minutes by rotating and
rocking the flask, during the last half hour they were allowed to
settle to the bottom.

Although the majority of the eggs settle to the bottom in ten
minutes, at the end of one-half hour there are always a few which,
on account of swelling or fragmentation, have failed to precipitate.
To prevent, these going over into the sample bottle and causing
an error due to absorption of iodine, Loeb placed filter paper over
the outlet. We found that a relatively hard filter paper was neces-
sary to retain all fragments of eggs, and that this interfered with
the rapid transfer of the solution. The error due to eggs in sus-
pension is negligible, especially since it was practically constant
in all of our experiments. For instance: a sample bottle filled with
water contained 8.09 parts per million of oxygen, while water from
the same jar run into a sample bottle in which had been placed
about one-hundred times as many eggs as the water from the deter-
mination flask, contained 7.85 parts per million, an error of 0.24
parts per million due to eggs in suspension. As in our experi-
ments, differences of 0.1—3.0 parts per million were obtained, an
error of 0.024 parts per million would not reverse the results.

When the eggs were fertilized or placed in parthenogenic solu-
tions they lost sufficient red pigment (MceMunn’s Echinochrome) to
color the water a straw yellow. In order to ascertain whether this
organic matter would vitiate the results, we took two sample bottles,
into one of which was placed a mass of elaeocytes containing about
fifty times as much echinochrome as is lost from the eggs in one
experiment, and syphoned into each tap water from the same
jar. ‘Tap water causes these cells to liberate their pigment. The
bottle containing water only, was found to hold 6.85 parts per
million of oxygen, while that contaminated by elaeocytes was
titrated as 6.36 parts per million of oxygen. We repeated this
using three sample bottles. Two of these filled with water gave

J. F. McClendon and P. H. Mitchell 467

6.86 and 6.91 parts per million respectively and the third contamin-
ated with elaeocytes liberating about one hundred times as much
pigment as is liberated in the experiments with eggs, gave 6.09
parts per million as the titration, showing an error of 0.8 parts per
million. Probably this loss was due chiefly to the broken up
cells, but if due entirely to the pigment, the error in our experi-
ments would be only .008 parts per million.

In experiment 3, the eggs were divided into two equal portions
and placed simultaneously in two determination flasks of equal
capacity. Therefore the eggs in the two solutions were in the same
stage of ripeness. In each of the other experiments the eggs were
all placed in one flask and treated successively with the various
solutions. In case of fertilized eggs Warburg observed! that the
oxidation rate rose steeply from fertilization to the 2-cell stage then
gradually to the 64-cell stage. In order to determine whether this
would be a great source of error on our experiments, we measured
the oxygen used by a mass of fertilized eggs in sea-water during
successive periods in the first six hours of development, and found
it to vary from 7.93 to 9.76 tenths of a milligram (Table 7). With-
in the duration of the majority of our experiments, however, the
variation was only from 7.93 to 8.96 tenths of a milligram or 11.5
per cent, and would be less between successive exposures. This
possible source of error would not reverse the results of the major-
ity of our experiments.

In making solutions of the same alkalinity as the sea water, a
colorimetric method was used, with phenolphthalein as indicator.
At the end of the season, the sea water was diluted with heavy
rains and failed to color phenolphthalein. The eggs behaved
abnormally, though whether this was due to the decrease in alka-
linity or salinity of the sea water or to some other cause was not
determined.

In making the eggs parthenogenetic with carbon dioxide, they
were placed with a small quantity of sea water in a “sparklet
syphon” and charged under slight pressure for about one minute.
At the end of five minutes they were poured into a large volume of
sea water and this was syphoned off and fresh sea water added.

1 Warburg: Zeitschr. f. physiol. Chem., lx, p. 448, 1909 and loc. cit.

468 Oxidation in the Sea Urchin’s Egg
RESULTS OF EXPERIMENTS.

i. Experiments with Unfertilized Eggs.

TABLE 1.

Oxidation in neutral van’t Hoff’s solution contrasted with that in neutral
NaCl solution.

| ia P| & a
&«§ Bet lie é a
hes Ho | 2& | ue p
EXPERIMENT SPLEEN So | a | cre o§ > nee
| p Ap Be Be ical
- sa | $2 | ge | ge MS
S ae 5a pa | i
| bed H a A, | o} |
. eae | ~
| ce °C min min a=
Neutral | |
First period. .| ; van’t 4 24 | 30—| 30 | 2.49
}
Hoff | | |
"| | | No ‘‘fertili-
: N l |.) Se NG Ee ran
Second Sets) S Eure \ 4 24 | 30°) 30-1) 2ona0| re
\* NaCl f | |) membrane
| | | formed.
}
1

TABLE 2.

Oxidation in neutral van’t Hoff’s solution contrasted with that in sea water
and alkaline NaCl solution.

Qe (=)
| iS B 5 S o 6 g E |
z bi 5 it | Es Zz Z |
EXPERIMENT | SOLUTION | gg | ees Ss Be ae REMARKS
USED | a g 8 = Be ee ele
ae] 2Bo| Sb Bae
fle a =) a °
| cc “1G; | min min ae |
Neutral | | | |
First period. .| 4 van’t | } 24°") 30 5) Sor azam
| Hoff. |
Second period Sea water | 24 | 30 30 |.2.65 ;
| _{ “Fertiliza-
+ NaCl ‘| | | || tion mem-
Third period {+—OH ? | | 24 30 30 | 3.12|4 branes’’ in
ions | | | | | very small
| per cent.
Fourth period Sea water | 24° | °30 30 | 3.48 | :
pe = "= ne by
y

J. F. McClendon and P. H. Mitchell

TABLE 3.

469

Oxidation in sea water contrasted with that in alkaline NaCl solution.

ae a a
| s | 8 | ce | ee] 8 |
EXPERIMENT saad a 2 e z z E 25 Zz | REMARKS
Bg | & = 3 a | £ a iM
S E 5 B 3
cc °c min min =
I. One-half
of eggs. .... Sea water 23 30 30 | 4.80 |
_ ( “Fertiliza-
Second half {¥ NaCl By eee
of eggs. ... . +-OH 23 | 30 | 30 | 6.90 ea”
ions | Trane
ima | | formed.
period.: 2... | Sea water 24.5| 35 | 25 |3.38|
“ NaCl | Bee se
. i ion mem-
Second period ; "OH / /24.5| 35 | 25 | 7.801) brane”
a) | | aa formed
Ill. First |
pered....3... | Seawater, 4.5, 24 30 30
“‘Fertiliza-
_{% NaCl tion mem-
Second period < +~OH 4.5 | 24 30 30 | 6.47 | branes” in
ions very small
per cent.

TABLE 4.

Oxidation in sea water contrasted with that in neutral NaCl.

|

a2 /8,/8,| 3
SOLUTION 5 E c 3 z 5 zZ :
EXPERIMENT © aan | g 2 & e e - z 5 z REMARKS
R os 5 I ee ao a
oF a5 p@ p2 *
> a a a So
ec °¢ min min a
1 eeirst
period...... | Bey water 5 23 30 30 | 2.65
(No “‘fertili-
i Neutral zation mem-
2 a7
Second period \ ™ NaCl 5 23 | 30 | 30 | 3.60 ae
| formed.
II. First
period. . | Sea water |2.3 | 23.5/ 30 | 30 | 1.22
Neutral ;
Second pro Nac] [| 2-3 23.5) 30 30 1.66 Ibid.

470

Effect of COs-parthenogenesis on oxidation.

Oxidation in the Sea Urchin’s Egg

TABLE 5.

Effect of fertilization.

)

ee | & B edexieok!
SOLUTION 6 e 3 : z : 2 | 2
EXPERIMENT AT 2 g B & c z E & | a | REMARKS
2a an | Ba Be RO
> a a a ; o |
° . 5 m
cc E min min am
I. First
period.....| Seawater | 4.5) 23.5) 30 30 4.58
ae | ( “Fertiliza-
After CO. | tion mem-
treatment. .| Sea water AV ay BBY 5 130) 30 6.00 branes” in
| | small per
cent.
II. First |
period...... | Seawater | 4.5) 24 | 30 | 30 4.11
AfterCO. | | | _ {Good mem-
treatment..| Seawater | 4.5 | 24 30 30 9.91 | brane
III. First | | formed.
period..... .. Sea water 25 30 30 2.52
After fertili- | Vs per cent of
zation......| Seawater | 5 | 25 | 30 | 30 | 12.94 eggs seg-
| | | ty mented.

SUMMARY.

1. The presence of ~OH ions in the medium, increases the rate
of oxidation in fertilized eggs of the sea urchin.

2. The oxidation rate of unfertilized eggs is increased by fer-
tilization or any treatment which causes them to develop partheno-
genetically.

In 1 and 2 we merely confirm and extend the observations of
Warburg.

3. Since it was shown by the senior author that fertilization or
parthenogenesis means increased ionic permeability of this egg, and
that the Fundulus egg, even after fertilization, is impermeable to
~Cl ions, the increase in permeability probably applies so far as the
anions are concerned, to "OH and ~HCO; or ~CO; ions. The car-
bonic acid anions are more concentrated within, and their outward
diffusion would cause a potential gradient which would pull other

J. F. McClendon and P. H. Mitchell A7I

IT. Experiments with Fertilized Eggs.

TABLE 6.
Oxidation in neutral van’t Hoff’s solution contrasted with that in sea water
and alkaline NaCl.
«Reg ae ea eee
3 Go | 22 | &z -
EXPERIMENT << se ad Zo ae | = z S = z REMARKS
aS mae <& <6 o
58 = E a) Fo
> & a a 2)
cc °¢ min min. mgr./10
I. First | Neutral Contained
peériod......| 4 van’t 3.6 | 23 30 30 6.47 some sea
Hoff water.
Second period) Sea water 3:6) 23 .| 30 30 7.43
Third ™ NaCl | |
period...... + OH | 3.6} 23 30 | 30 | 12.28
Fourth es | Very few
period ....| Seawater; 3.6 | 23 30 30 5.97 eggs dead.
(E hed
Il. First Neutral } eer
period...... van’t Nag: 23 30 30 | 3.91 Hoff sol
:
Hoff and ferti-
| | lized in it.
Second period Seawater 4 | 23 30 30 oat
™ NaCl ) |
Third period |;+~OH?; 4 23 30 30 7.53
ions

Fourth Period! Seawater | 4 | 23 | 30 30 6.27

anions, hence —OH ions, into the egg, thereby increasing the inter-
nal concentration of -OH ions. This increase in ~OH ions proba-
bly causes the increased oxidation.

4. The increase of —OH ions in the medium causes even in
unfertilized eggs, an increased oxidation. This is not interpreted
as indicating that the unfertilized egg is normally permeable to
—-OH ions, but that increased alkalinity causes increased permea-
bility.

5. Increase in permeability is a gradual process. Beginning
with the relatively impermeable unfertilized egg, and denoting
degree of permeability by numerals, we have the following series,

472 Oxidation in the Sea Urchin’s Egg

TABLE 7.

Effect of alkaline NaCl on oxidation siz hours after fertilization.

| Pere F
| s | BE | oe [Se] 8
ae | SOLUTION Sas 51) OSe| oa a
EXPERIMENT USED 2 g 2 | 5 z | g E 8 REMARKS
ee) g2 | ge | ga | &
> is) =) a °
cc | 6 min min *
| Thirteen
Uy v So | 0 minutes
First period. .; Sea water aad 22 | 20 3 8.90 after ferns
: | | | lization.
Second period Seawater | 7 | 22 | 20 | 30 8.96) 2-cell stage.
Third period | Seawater | 7 | 22 | 20 | 30 | 8.33) 4-cell stage.
Fourth period, Seawater | 7 | 22 | 20 | 30 | 7.93) 16-cell stage.
Fifth period..; Seawater | 7 | 22 | 20 | 30 | 8.18) 32-cell stage.
Sixth period... Seawater | 7 22 20 30 9.76, Many retarded.
Seventh Aa NaCl | M bl
period..... \¥ ++ -OH }). 7 | 22 | 20 | *30. \eiaii7a eee
; | | tulae.
ions J] |
Eighth period Seawater | 7 | 22 | 20 | 30 | 8.98 All alive.

I. slightly increased oxidation, IJ. greater increase in oxidation
rate, and imperfect “fertilization membrane” formation, III. oxi-
dation still further increased, membrane formation perfect, fol-
lowed by segmentation of the egg, IV, ditto except that oxidation
is still further increased, and the eggs die sooner or later if oxida-
tion is not reduced, V. oxidation enormous, membrane formation
but no segmentation, premature death.

It is supposed that the primary effect of many toxic substances
is an abnormal increase in the permeability of the egg, and ferti-
lized eggs are more susceptible because they are already more per-
meable than unfertilized eggs.

ty
ae ;
a

A Note on the Dynamics of Cell Division.
A Reply to RoBERTSON
by
J. F. McClendon.

(From the Embryological Laboratory of Cornell University
Medical College, New York City.)

With 2 figures in text.

Eingegangen am 15. November 1911.

In a recent paper ROBERTSON!) attempts to strengthen his hypo-
thesis of cell division put forward previously, which is as follows:
In the polar synthesis of nuclein from lecithin, cholin is formed as
a by-product, and diffusing in all directions, reaches a maximum
concentration at the equator, where it (or a soap formed from it)
diminishes the surface tension and leads to constriction of the cell.
This hypothesis is erroneous for two reasons:

First: There occurs no appreciable synthesis of nuclein during
cleavage. Masine@?) and recently SHACKELLS) have shown that there
is as much nuclein in the egg at the 2 cell stage as during the blas-
tula stage.

Second: The surface tension is not diminished, but is relatively
increased at the equator as shown in a model of cell division des-
eribed below. Following the preliminary experiments of Gap,
QuINcKE‘) observed that if a drop of rancid olive oil in water is
touched with an alkaline solution, even with such a slightly alkaline

1) Roperrson, T. B., Arch. f. Entw.-Mech. 1911. XXXII. S. 308.

2) MasinG, Zeitschr. Physiol. Chem. 1910. LXXVII. S. 161.

3) SHACKELL, Science. 1911. N.S. XXXIV. p. 573.

4) QuINCKE, Sitzungsber. d. kgl. preuB. Akad. d. Wiss. zu Berlin. 1880.
XXXIV. S. 791.

264 J. F. McClendon

solution as egg white, the surface touched decreases in tension,
spreads and is pushed forward. He explained that this was due to
soap formation. He supposed that living cells were covered with a
thin layer of oil, and movements were due to local formation of soap.

These experiments were continued by Birscuxi!) who filled the
drop of olive oil with globules of an alkaline solution. When one
of these globules burst, the alkaline solution spread over the adjacent
surface of the oil drop and locally lowered its surface tension, causing
a protuberance. BirscuLi supposed the movements of Amoeba to
be of similar origin.

BERNSTEIN?) described surface tension movements in drops of
mercury, which are now well known to the majority of biologists.

Similar movements were seen by JENNINGS?) in drops of clove oil.

Fig. 1.

In all of the above mentioned experiments a decrease in surface
tension is shown to cause a protrusion of the surface. ROBERTSON,
however, claims that a decrease of surface tension causes a receding
of the surface, and when the decrease is along an equator, the drop
is cut in two. He used a drop of 2 parts chloroform and 3 parts
rancid olive oil and immersed it in water. When a thread soaked
in n/10 NaOH was laid across the drop, the latter was cut in two,
as he supposes, by the decrease in surface tension following soap
formation. Contrary to Roprerrson IJ have failed utterly to obtain
a division of the drop by this method. When the NaOH has
diffused over the whole surface, a flattening of the drop occurs, but
a constriction never takes place.

1) BUTSCHLI, Quart. Journ. Micr. Sc. 1890. XXXI. p. 99.
*) BERNSTEIN, Arch. f. d. ges. Physiol. 1900. LXXX. S. 628.
3) Jenninas, Journ. Appli. Micr. and Lab. Methods. 1902. V. p. 1597.

The result is very different when the NaOH is applied to the .

A Note on the Dynamics of Cell Division. : 265

poles of the drop. Fig. 1 represents a drop of rancid oil and chloro-
form submerged in water. n/10 NaOH is applied to the poles’by
means of the two pipettes. If the NaOH is applied to the two
poles at exactly the same time and rate, the drop constricts
as in Fig. 1, and divides in two.

The details of this process are shown in Fig. 2. When the sur-
face tension is decreased at the poles, these surfaces stretch because
the tension is overbalanced by the greater surface tension at the
equator. This causes a surface movement from the poles to the
equator as shown by the dark arrows. The tension of the surface
of the drop causes hydrostatic pressure in the interior. The surface
tension, being diminished at the poles, is overbalanced by the hydro-
static pressure and the surface is protruded. This causes internal
currents from the equator toward the poles of the drop, as shown
by the dashed arrows.

Fig. 2.

This model of cell division simulates the division of an Amoeba
more than it does that of an egg or tissue cell. In these latter, the
presence of a membrane or adjacent cells prevent the daughter cells
from moving apart and the dumb bell shape is not formed.

The mechanics of cell division may be illustrated by a more
tangible model. A rubber balloon is inflated with air and attains a
spherical shape. The rubber may represent the surface film, and
with uniform thickness of rubber we obtain uniform tension and
spherical shape. If the equator of the balloon is re-inforced with a
rubber band, the tension along the equator is increased and the
balloon is constricted equatorially. The rubber band may be cemented
to the balloon before it is inflated or the balloon originally made
thicker along the equator, and in every case the result is the same.

The division of a mercury drop, due to decrease in surface
tension at the poles following electric polarization in these regions,
as observed by CurISTIANSEN!) is described in FreuNDLICH’s Capillar-

!) CHRISTIANSEN, Drud. Ann. 1903. XII. p. 1072.

266 J. F. McClendon, A Note on the Dynamics of Cell Division.

chemie, p. 212—5. The mercury drop in KNOg solution becomes
positively charged. Where the positive current enters, the drop
becomes less. positive, i. e. the polarization decreases, and the surface
tension increases. At the opposite pole the surface tension decreases.
As the current increases, the pole at which the positive current enters,
passes the neutral point and becomes negative, leading to a decrease
in surface tension. We have then the surface tension decreased at
one pole more than at the other. One pole is strongly positive and
the other slightly negative, and the neutral region forms a band
around the drop nearer to the negative pole than to the positive.
This neutral band is the region of greatest surface tension. The drop
finally divides near the neutral region.

The reason the drop does not divide exactly in the center of
the neutral region, or region of greatest surface tension, may be that
surface currents hinder such a division. However, this is a detail
which need not be essential to our main topic, since the consistency
of mercury is very different from that of protoplasm.

Summary.

If a drop of rancid oil and chloroform is immersed in water and
n/10 NaOH solution is allowed to diffuse against two opposite poles
of the drop at the same time and rate, the drop constricts and divides
in two along the equator.

This division is due to decrease in surface tension following soap
formation at the poles, or relative increase in surface tension at the
equator. Contrary to ROBERTSON, a decrease in surface tension along
the equator does not divide a drop.

Zusammenfassung,

Gibt man einen Tropfen ranziges Ol mit Chloroform in Wasser und laBt
‘/;>-Normal-Natronlauge gegen zwei entgegengesetzte Pole des Tropfens gleich-
zeitig und gleichmiGig diffundieren, so zieht sich der Tropfen zusammen und
unterliegt einer Zweiteilung entlang seinem Aquator.

Diese Teilung ist bedingt durch eine Verminderung der Oberfliichenspan-
nung, welche auf die Seifenbildung an den Polen folgt, oder durch eine relative
Vermehrung der Oberflichenspannung am Aquator. Im Gegensatz zu ROBERTSON
fiihrt eine Abnahme der Oberflichenspannung entlang dem Aquator nicht zur

Tropfenteilung. (Ubersetzt von W. Gebhardt.)

TE

|)
=~]
bed

(From the Histological Laboratory of Cornell University Medical College,
New York City.)

Ein Versuch,
améboide Bewegung als Folgeerscheinung
des wechselnden elektrischen Polarisations-
zustandes der Plasmahaut zu erklaren.

Von
J. EF. McClendon.

(Mit 4 Textfiguren.)

Nach der Ansicht vieler Autoren werden amdboide Bewegungen
durch Anderungen der Spannung der Oberfliche hervorgerufen.
Dieser Spannungszustand der Oberfliche kann entweder eine echte
Oberflachenspannung oder, nach Rhum bler*) zuweilen eine Spannung
des Ektoplasmas als Folge von Wasserverlust (,,Gelatinierungsspannung ~
oder Gelatinierungsdruck*) sein. In dieser Mitteilung soll nur von
echter Oberflichenspannung die Rede sein.

Lasst man Kaliumbichromat gegen einen Tropfen Quecksilber

in verdiinnter Salpetersiure diffundieren, dann sendet der Queck-
- silbertropfen von der Stelle, die zuerst mit dem K,CrO, in Be-
rihrung tritt, einen amdboiden Fortsatz aus. Es findet namlich
unter Verminderung der Oberflichenspannung eine Bewegung der
fusseren Schichten von dem Bichromat weg und eine solche der
inneren gegen das letztere zu statt. Ahnliche Strémungen wurden
bei der Bewegung vieler Amében beobachtet. Hier verursachen die
Riickstrémungen an der Oberfliche und die Vorwartsstrémuagen im
Inneren naturgemiss einen allmihlichen Austausch von Ekto- und
Endoplasma. Dieser Vorgang, den Rhumbler den ,,Ektoendoplasma-

1) L. Rhumbler, Zur Theorie der Oberflichenkrafte der Amében. Zeitschr.

f. wissensch. Zool. Bd. 88. S. 1. 1905.
18**

212 J. F. McClendon:

prozess nennt, ist schon vor Jahren von Wallich und Montgomery ‘)
(1979, 1881) beobachtet worden.

Nach Jennings?) findet ein derartiges Abfliessen der vor-
geschobenen Oberfliche nicht bei allen Amdben statt. Doch liesse
sich dessen Ausbleiben in solchen Fallen durch die Annahme einer
ziheren Plasmahaut und Alveolarwandsubstanz des Protoplasmas
geniigend erkliren. Das oberflachliche Abfliessen ware dann teilweise
auf die einzelnen Alveolen beschrinkt. Rhumbler jedoch erklirt
dieses Ausbleiben der oberflachlichen Str6mungen auf Grund von
3erthold’s Theorie dahin, dass die Fortbewegung der Amébe durch
einseitiges Anhaften an der Unterlage hervorgerufen wird.

Zweifellos hat die Plasmahaut einen Einfluss auf den Mechanis-
mus der Bewegung. Nach Quinecke ist dieselbe ein Fett-, nach
Overton ein lipoides Hautechen, doch ist ihre wirkliche Zusammen-
setzung noch nicht sichergestellt. Sie ist in Wasser unloslich, je-
doch fiir Wasser und andere, namentlich lipoidlésliche Substanzen
durchlissig. Das Cytoplasma lebender Zellen oder Kier enthilt
Lipoideiweissverbindungen, die in Wasser nicht ldéslich sind, sich
aber bei Beriithrung mit Wasser unter Freiwerden von Lipoiden zu
zersetzen scheinen [McClendon®), 1910]. Nach dem Gibbs’ schen
Prinzip sammeln sich diese Lipoide an der Oberfliche und bilden
eine Schicht, die eine weitere Zersetzung der Lipoideiweisverbindungen
verhindert. Dieses lipoide Hautchen ist unter gewohnlichen Um-
stiinden von ultramikroskopischer Dicke; wird hingegen dem Wasser
etwas Alkohol zugefiigt, so kann unter Umstinden ein verhaltnis-
missig dickes Hautchen auf der Oberflaiche (z. B. von Hihnerei-
dotter) geformt werden.

Dass die Plasmahaut der Amoébe fir Anionen weniger durchlassig
zu sein scheint als fir Kationen, ist eine Vermutung, die durch zwei
Tatsachen nahegelegt wird:

1) E. Montgomery, Elementary Functions and Primitive Organization of —
Protoplasm. St. Thos. Hos. Repts., London 1878, n. s. IX. 1878. — Zur Lehre
von der Muskelkontraktion. II. Die améboide Bewegung. Pfliger’s Arch.
Bd. 25 S, 499.

2) H. S. Jennings, Contributions to the Study of the Behavior of Lower
Organisms. VI, Publ. Carnegie Instit. Washington Nr. 16 p. 129. 1904.

3) J. F. McClendon, Dynamics of Cell Division. I]. Americ. Journ:
of Physiol. vol. 27 p. 240. 1910.

Ein Versuch, améboide Bewegung als Folgeerscheinung etc. 973

1. Wird durch einen Wassertropfen, in dem eine Amdbe
schwebt, ein schwacher elektrischer Strom geleitet, so wandert das
Tier passiv gegen die Anode.

2. Wird ein starker elektrischer Strom durch das Wasser ge-
leitet, dann beginnt der Zerfall der Amébe am anodalen Pol.

Der zerstérende Einfluss eines elektrischen Stromes auf lebendes
Gewebe ist vielleicht die Folge einer Anhaiufung von Ionen, die in
ihrer freien Bewegung durch gewisse Gewebsstrukturen behindert
werden. Da nun an der Amobe die Zerstérungserscheinungen zuerst
an der Oberflache auftreten, so diirfte die Plasmahaut fiir eine Be-
hinderung der Jonenwanderung verantwortlich zu machen sein; die
Ionen wiirden sich, da sie die Plasmahaut nicht leicht passieren
koénnen, hinter derselben anhaufen, und diese Anhaiufung wide die
sichtbaren Erscheinungen des Zerfalles hervorrufen.

Fig. 1. Schematische Darstellung des Einflusses eines elektrischen Stromes auf

eine Amébe. A= Anode. AK = Kathode. Der grosse Kreis stellt die Plasma-

haut dar, die kleinen Kreise Ionen mit ihrer entsprechenden Ladung. Die
Pfeile deuten die Bewegungsrichtung der Ionen an.

Der Zerfall der oberflachlichen Schichten tritt nicht an beiden
Polen der Amdobe gleichzeitig auf, sondern zunichst nur an der
Anode, so dass es den Anschein hat, als ob die Plasmahaut der
Durchwanderung der Anionen einen grésseren Widerstand entgegen-
setzt als der der Kationen. Die Fig. 1 stellt diese Annahme
schematisch dar. Der grosse Kreis reprisentiert die Plasmahaut, die
kleineren die Ionen, deren Ladung durch + oder — bezeichnet ist.
Die Bewegungsrichtung der Ionen ist durch Pfeile angedeutet. Die
positiven Ionen scheinen die Amébe ungehindert zu passieren, wahrend
die negativen ausserhalb des Zelleibes denselben leicht umgehen
kénnen. Jene negativen Ionen jedoch, die im Protoplasma ein-
gesperrt sind, scheinen die Oberhaut nicht passieren zu kOnnen,
stauen sich daher hinter derselben und rufen dann die gewissen
Auflésungserscheinungen hervor.

74 J. F. McClendon:

Wird jedoch ein Strom, der zu schwach ist, um das Plasma zu
zerstoren, durch das Wasser geleitet, dann wandert die Amdbe passiv
gegen die Anode, eine Beobachtung, die Hirschfeld’s*) (1909) An-
nahme einer positiven Ladung derselben nicht bestatigt. Diese
passive Wanderung der Amoébe koénate in folgender Weise erklirt
werden. Im Innern derselben wird ununterbrochen ein Elektrolyt .
erzeugt, dessen Anionen die Plasmahaut nicht passieren kénnen.
Diese geben der Amodbe eine negative Ladung, so dass das Tier,
wenn ein Strom durch das Wasser geleitet wird, gegen die Anode
gezogen wird.

Da unter allen Elektrolyten, die unter solechen Umstanden er-
zeugt werden kénuten, die Kohlensiure wohl der ausgiebigste ist,
dirfte deren Gegenwart den oben gestellten Forderungen genigen.

Fig. 2. Schematische Darstellung des elektrischen Polarisationszustandes

der Amédbenoberflache als Resultat einer relativen Undurchlassigkeit der

Plasmahaut fur die Anionen der Kohlensiéure. Zeichen wie in Fig. 1.
Ks ist allerdings wahr, dass das elektrolytische Dissoziationsvermégen
der Kohlensiure sehr gering ist. Dies wiirde dann eben eine starke
Konzentration derselben noétig machen, die méglicherweise in der
Zelle existiert. Wir kénnen nun annehmen, dass die H+-Ionen aus
der Amébe herauswandern kénnen, wihrend die HCO,- und CO, -
Ionen innerhalb derselben zuriickgehalten werden und ihr die ndétige
negative Ladung geben. Diese Scheidung der Ionen wirde noch durch
die gréssere Schnelligkeit, mit der die H+-Ionen durch das Plasma
wandern, gefordert. Die Anionen im Inneren und jene Kationen,

die durch sie auf der Oberflache zuriickgehalten werden (Fig. 2), —

bewirken eine Polarisation der letzteren, ahnlich dem Lippmann-
schen Phanomen im Kapillarelektrometer. Infolge dieser Polarisation
muss die Oberflichenspannung eine geringe sein, wie es tatsachlich

1) L. Hirschfeld, Ein Versuch, einige Lebenserscheinungen der Amében
physikalisch-chemisch zu erkliren. Zeitschr. f. allgem. Physiol. Bd. 9 S. 529. 1909.

Ein Versuch, améboide Bewegung als Folgeerscheinung etc. 275

die Beobachtung lehrt. Obzwar die Oberfliche einer Amébe még-
licherweise aus Lipoiden besteht, besitzt das Tier eine viel geringere
Oberflichenspannung als ein Oltropfen im Wasser.

Unter solchen Umstinden wiirden amédboide Bewegungen durch
értliche Verainderungen der Obertlichenspannung erzeugt, die ihrer-
seits eine Folge der wechselnden Oberfliichenpolarisation sind.
Wahrend die CO,-Ionen vielleicht nicht leicht passieren kénnen
und die Kationen unter ihrem Einflusse zum Teil zuriickgehalten
werden, kénnten andererseits die nicht dissoziierten Molekiile als
CO, auswandern, da CO, in Lipoiden léslich ist. Der Polarisations-
zustand kénnte dann nur so lange aufrechterhalten werden, als CO,
innerhalb der Amébe in gleichem Maasse erzeugt wird, als sie aus-
wandert. Wiirde an irgendeiner Stelle die Kohlensaurebildung
steigen oder fallen, dann wiirde die gleichzeitige Polarisations-
verinderung des nichstliegenden Teiles der Oberflache spontane Be-
wegung erzeugen. Die Kohiensiurebildung wird vielleicht zeitweise
durch fussere Bedingungen beeinflusst, z. B. durch Substanzen,
die von anderen Organismen in das Wasser abgegeben werden. Eine
Erhéhung der CO,-Bildung wiirde dann positiven Tropismus hervor-
bringen.

Andererseits wiirde eine Erhéhung der Durchlissigkeit der
Plasmahaut eine Herabsetzung der Polarisation derselben zur Folge
haben. In einer fritheren Arbeit (1910) habe ich eine Reihe von
Agentien aufgezihlt, die die Durchlassigkeit der Plasmahaut fir
Anionen erhéhen. Dieselben Agentien bewirken auch in der Amédbe
eine Erhéhung der Oberflichenspannung, die sich in einer grésseren
Abrundung des Korpers aussert. Wird die Amébe nur auf einer
Seite von diesen Agentien beeinflusst, dann zeigt sie negativen
Tropismus. Negativer Tropismus kann dadurch erklirt werden, dass
die ihn bewirkenden mechanischen, thermischen, chemischen u. dgl.
Kinfliisse die Plasmahaut an der der Einwirkung nichstgelegenen
Stelle auflockern; der daraus resultierende Verlust an Polarisation
unter gleichzeitiger Erhéhung der Oberfliichenspannung veranlasst
die Amébe, sich von dem schiadlichen Einfluss zuriickzuziehen.
Der Teil der Oberfliche, an dem die Plasmahaut verindert
_worden ist, bleibt aber nicht depolarisiert, da er seine frihere Un-
durchlassigkeit zuriickerhalt, sobald sich die Amébe dem schidigenden
Kinfluss entzogen hat.

Die Polarisation ist nicht der einzige Faktor, der die Ober-

276 J. F. McClendon:

flichenspannung zu verindern vermag. Ks ist z. B. ee bekannte
Tatsache, dass Seifen die Oberflichenspannung an Lipoiden ver-
mindern.- Wenn nun die Plasmahaut aus Lipoiden besteht, sollte man
[mit Bitsehli, J. Loeb, Robertson, Michaelis u. a.] ver-
muten, dass Seifen eine Herabminderung der Oberflichenspannung
an der lebenden Zelle bewirken; doch habe ich das gerade Gegen-
teil beobachtet. Eine Kapillarréhre wurde mit fester Seife oder
Seifenlésungen von verschiedener Konzentration gefiillt und mittels
der ,mechanischen Hand“ [MceClendon?), 1909] so weit in Wasser
eingetaucht, bis ihre feine Offnung ganz in die Nahe einer grossen
Amoeba proteus kam. Das Tier zeigte immer deutlichen nega-
tiven Chemotropismus, in dem es rasch von der Seife, die aus der
Kapillarréhre heraussickerte, wegwanderte. Manchmal war die Be-
weeung nicht rasch genug, um das Leben des Tieres zu retten.
Leider konnte der genaue Zeitpunkt, an dem die ersten Seifen-
molekiile die Amoébe erreichten, nicht bestimmt werden. So liess
sich daher auch nicht feststellen, ob ein kurzdauernder Vorstoss des
Tieres gegen die Seifenlésung zu, wie er manchmal beobachtet wurde,
als eine ,,Reiz-“ oder spontane Bewegung aufzufassen sei. Auf jeden
Fall kénnen wir schliessen, dass der hauptsachlichste Einfluss, den
die Seife auf die Amébe ausiibt, geeignet ist, die Durchlissigkeit der
Oberfliche fir Anionen zu erhéhen, da das Tier negativen Tropis-
mus zeigt.

In einer kiirzlich erschienenen interessanten Arbeit tiber die
Rolle der verschiedenen Kolloidalzustande der Oberflache der Amében
bei Nahrungsaufnahme kam Rhumbler®*) (1910) zu dem Schlusse,
dass die Annahme einer festen, elastischen und relativ dicken Ober-
flichenschicht notwendig ist, um jene Form der Nahrungsaufnahme
zu erkliren, dass er ,Circumvallation“® nennt. Er vermutet, dass
,Circumvallation* durch eine értliche Auflésung der halbfesten Ober-

1) J. F. McClendon, Protozoan Studies I. Reactions of Amoeba Proteus
to Minutely Localized Stimuli. Journ. of exper. Zool. vol. 6 p. 265. —
Autoreferat iiber vorstehende Abhandiung in Arch. f. Entwicklungsmechanik
Bd. 57 5. 323. 1909. — The Reaction of Amoeba to Stimuli of Small Area. —
Americ. Journ. Physiol. vol. 21. 1908. Proc. Americ. Physiol. Soc. 1907 p. 13. —

2) L. Rhumbler, Die verschiedenartigen Nahrungsaufnahmen bei Amoben
als Folge verschiedener Kolloidalzustiande ihrer Oberflichen. Areh. f. Entwicklungs-
mechanik Bd. 30 8. 194. 1910.

Ein Versuch, amboide B: wegung als Folgeerscheinung etc. 277

flichenschicht durch einen von der Beute ausgeiibten ,Reiz“ ein-
geleitet wird ').

Es gibt jedoch eine Art der Nahrungsaufnahme, die der ,,Cireum-
vallation* zwar verwandt, aber in folgender Weise durch Ver-
anderungen der Oberflichenspannung erklirlich ist: wenn z. b. eine

Fig. 3. Schematische Darstellung der Nahrungsaufnahme einer Amébe. Die
Pfeile deuten die Richtung an, in der eine Erhéhung der Oberflachenspannung
erfolgt. Der kleine Kreis stellt eine Algenzelle dar. g—k — Querschnitte durch

den Plasmaring der Figuren e—f.

Amoeba proteus sich in der Nihe einer Algenzelle befindet
(Fig. 3a), dann bewirken auf Grund meiner friiheren Voraussetzungen

1) Ks ist zweifellos, dass eine feste Oberflaiche die Nahrungsaufnahme der
Amébe nicht verhindert. Schaudinn (1899) (Generationswechsel von Tricho-
sphaerium Sieboldi. Anhang d. Abhandl. Berliner Akad. d. Wissensch. 1899
S. 1) beobachtete die Aufnahme von fester Nahrung bei Trichosphaerium
Sieboldi, dessen Koérper von einer Gallerthiille umgeben ist, in die zahlreiche
radiaér gestellte Stibchen, MgCO;, eingebettet sind. Eine zum mindest halbstarre
Oberfliichenschicht ist, wie Rhumbler beweist, sogar notwendig, um die
Nahrungsaufnahme durch ,Invagination* zu erméglichen.

278 J. F. McClendon:

Stoffe, die aus der Zelle ausgeschieden werden, eine Steigerung der
CO,-Erzeugung im niichstliegenden Teil des Amébenleibes; denn die
Ambébe breitet sich gegen die Alge aus. Manchmal rollt sie die
letztere sogar etwas vor sich her (Fig. 36). Die Berithrung mit der
Alge oder die starkere Konzentration der reizenden Substanzen ver-
ursacht eine lokale Erhohung der Durehlissigkeit und der Spannung
eines Teiles der Plasmahaut, welcher das Feld verminderter Spannung
durehschneidet (Fig. 3c). Die seitlichen Partien schieben sich nun
weiter nach vorne, bis sie die Beute ganz umgeben (Fig. 3d) und sich
dann vor derselben vereinigen (Fig. 3e). Die Beute ist dann von
einem Protoplasmaring eingeschlossen. Die Oberflachenspannung be-
strebt nun eine Verkleinerung des letzteren. Manchmal kann der
dabei von allen Seiten einwirkende Druck ein Herauspressen der
3eute nach oben zur Folge haben, so dass die Nahrungsaufnahme
misslingt. Unter giinstigen Bedingungen hingegen schliesst sich der
Ring tber und unter dem Fremdkoérper, wie es im Querschnitt in
Fig. 3g und h dargestellt ist; gleichzeitig wird auch etwas Wasser
mit der Beute eingeschlossen, und zwar dirfte, wenn die Amébe an
der Unterlage anhaftet, etwas mehr Wasser eingeschlossen werden.

Diese Methode der Nahrungsaufnahme nimmt eine Zwischen-
stellung zwischen Rhumbler’s Klassen ,,Cireumfluenz* und ,,Cireum-
vallation“ ein, indem die Amobe in Beriihrung mit der Beute kommt,
zugleich aber Wasser mit der Nahrung aufnimmt.

Versuche, die ich an Seeigeleiern unternommen habe, unter-
stiitzen meine Ausfiihrungen. Dass Kier améboide Beweeungen aus-
fihren kénnen, ist eine bekannte Tatsache. Prowazek?) (1903)
beobachtete an Seeigeleiern, die in die Kérperflissigkeit einer
Annelide gelegt wurden, aktive Bewegungen; ein Ei nahm eine
»Hlaocyte“ in sich auf.

Wenn ein allmihlich stirker werdender elektrischer Strom durch
eine isotonische Zuckerlésung geleitet wird, die Seeigeleier enthalt,
sO muss man eine Erhéhung der Polarisation in dem der Anode ~
nachstgelegenen Teile der Plasmahaut erwarten, da die Anionen, die
hinter der Membran zurickgehalten werden, eine Anhaiufung der
Kationen an der Aussenseite hervorrufen (Fig. 4a). Die Anionen

1) Prowazek, Studien zur Biologie der Zelle. Zeitschr. f. allg. Physiol.
Bd§2 S. 385. 1903. ;

Ein Versuch, améboide Bewegung als Fo!geerscheinung: ete. 279

des Wassers kénnen das Ei umgehen, wihrend die Kationen es
durchwandern. Die Erhéhung der Polarisation muss eine Herab-
setzung der Oberflichenspannung und damit eine Ausdehnung der
Eioberflache gegen die Anode zu veranlassen. Zahlreiche Beobach-
tungen bestatigten diese Vermutung, indem das Ei unter den ge-
stellten Bedingungen jedesmal ein oder mehrere Pseudopodien gegen
die Anode ausstreckte. In einigen Fallen wurde auch ein Abfliessen
der oberflichlichen Schichten bemerkt.

Das beschriebene Phinomen war von sehr kurzer Dauer, da das
vorgestreckte Cytoplasma bald zu desintegrieren begann und osmo-

Fig. 4. Schematischer Versuch, den Tropismus der Amébe gegen die Kathode
zu erklaren. Zeichen wie in Fig. 1. (In der Amébe Aussert sich Galvano-
tropismus an der Kathode, Kataphorese an der Anode.)

tische Erscheinungen die Bewegungen auf Grund der Spannungs-
differenzen verhillten. Der Zersetzungsprozess wurde wahrscheinlieh
durch die Anhiufung der Anionen eingeleitet.

Wihrend in einem schwachen Strom das Seeigelei Pseudopodien
gegen die Anode ausstreckt, zeigt die Amébe Tropismus nach der
Kathode. Kin starker Strom aber verursacht in beiden Formen eine
Zersetzung am anodalen Pol. Diese Tatsache scheint anzudeuten,
dass die Verminderung der Oberflichenspannung und der Zerfall des
Plasmas im Seeigelei durch ahnliche, in der Amébe aber dureh ent-
gegengesetzte Faktoren hervorgerufen werden. Doch kann dieser
anscheinende Widerspruch dadurch erkliirt werden, dass auch in der

Amdbe die Polarisation des anodalen Poles steigt; bevor jedoch ge-
Pfliger’s Archiy fiir Physiologie. Bd. 140. 19

280 J. F. McClendon: Kin Versuch, améboide Bewegung ete.

niigend Zeit verstreicht, um eine Bewegung auszufiihren, wird die
Plasmahaut zerstért, so dass der Polarisationsgrad dieser Stelle unter
das Normale der tibrigen Oberflache sinkt (Fig. 40). Der Schluss-
effekt des Stromes wire somit verminderte Polarisation der anodalen
Gegend, und die Amébe wandert gegen die Kathode.

' Die erhdhte Dureblassigkeit eines Teiles der Plasmahaut wiirde
die Polarisation der gesamten Oberfliche etwas herabsetzen, da die
Anionen jenen weniger dichten Teil durchwandern kénnten. Doeh
wird in der Amébe ununterbrochen CO, erzeugt und wahrscheinlich
um so energischer, je schneller sie weggeschafft wird, wie es ja bei
allen Endprodukten chemischer Vorginge der Fall ist. Aus diesem
Grunde dirfte die Polarisation des nicht desintegrierenden Teiles
der Oberfliche sehr wenig, wenn iiberhaupt, herabgesetzt werden.

Der -Potentialabfall zwischen den beiden Seiten jenes Teiles der
Plasmahaut, der der Kathode zunichst liegt, ist entgegengesetzt dem
Potentialabfall, der an jener Stelle von den Elektroden erzeugt
wirde (Fig. 40). Man muss daher annehmen, dass die normale
Polarisation der Plasmahaut einem Potentialabfall entspricht, der
stirker ist als der des umgebenden Wassers, da sonst der erstere
durch den letzteren unterdriickt wiirde.

POU BLACA TTORMS

OF

Cornell University

Meee Al, COLLEGE

@:

eet) / fF S

FROM THE

Department of Anatomy
@&

VOLUME iil

| 1912
. we ee oF OR K CEE

jby,

6.

-1

CONTENTS

Being reprints of studies published in 1912.

. AN EXPERIMENTAL STUDY OF RACIAL DEGENERATION IN

MAMMALS TREATED WITH ALCOHOL.
By Charles R. Stockard.
Archives of Internal Medicine, Vol. X, 369-398 and 5 figures.

- IS THE CONTROL OF EMBRYONIC DEVELOPMENT A PRAC-

TICAL PROBLEM? By Charles R. Stockard.
Proceedings Am. Philosophical Soc., Vol. LI.

. AN EXPERIMENTAL STUDY OF THE INFLUENCE OF ALCO-

HOL ON THE GERM CELLS AND THE DEVELOPING

EMBRYOS OF MAMMALS.
By Charles R. Stockard and Dorothy M. Craig.
Archiv f. Entwicklungsmechanik, Vel. XXXV, 569-584.

. FUTTERUNGSVERSUCHE AN AMPHIBIENLARVEN.

By J. F. Gudernatsch.
Zentralblatte f. Physiologie, Vol. XXVI, No. 7

PoeewING. EXPERIMENTS ON TADPOLES. I. THE INFLU-

ENCE OF SPECIFIC ORGANS GIVEN AS FOOD ON
GROWTH AND DIFFERENTIATION. A CONTRIBU-
TION TO THE KNOWLEDGE OF ORGANS WITH IN-

TERNAL SECRETION. By J. F. Gudernatsch.
Archiy f. Entwicklungsmechanik, Vol. XXXV, 457-483 and 1 plate.

THE BEHAVIOR AND RELATION OF LIVING CONNECTIVE
TISSUE CELLS IN THE FINS OF FISH EMBRYOS
WITH SPECIAL REFERENCE TO THE HISTOGENESIS
OF THE COLLAGINOUS OR WHITE FIBERS.

By Jeremiah S. Ferguson.
Am. Jour. of Anatomy, Vol. XIII, 129-143 and 4 plates.

. THE RELATION OF MUSCLE CELL TO MUSCLE FIBRE IN

VOLUNTARY STRIPED MUSCLE. By W. M. Baldwin.

Zeitschrift f. allgem. Physiologie, Vol. XIV, 130-145 and 2 plates.

. THE RELATION OF THE SARCOLEMMA TO THE MUSCLE

CELLS OF. VOLUNTARY VERTEBRATE STRIPED
MUSCLE FIBRES AND ITS MORPHOLOGICAL NA-
TURE. By W. M. Baldwin.

Zeitschrift f. allgem. Physiologie, Vol. XIV, 146-160 and 1 plate.

10.

16.

THE RELATION OF MUSCLE FIBR@LLA TO TENDON FIBRE

LZ IN VOLUNTARY STRIPED MUSCLES OF VERTE-
BRATES. By W. M. Baldwin.

Morphologisches Jahrbuch, Vol. XLV, 249-266 and 1 plate.

MUSCLE FIBRES AND MUSCLE CELLS OF THEA.

WHITE MOUSE HEART.
Anatomischer Anzeiger, Vol. 42, 177-181 and 2 figures.

By W. M. Baldwin.

. DIE ENTWICKLUNG DER FASERN DER ZONULASZINNIT IM
AUGE DER WEISSEN MAUS NACH DER GEBURT.

_By W. M. Baldwin.

Archiv f. mikroskop. Anatomie, Vol. LXXX, 274-305 and 2 plates. ~

..y

CELLS UN VEER:
- Science, N. S., Vol. XXXVI, 90-92.

2 RHYTHMICAL ACTIVITY OF ISOLATED HEART SMiUSerE
By Montrose T. Burrows.

3. RHYTHMISCHE KONTRAKTIONEN DER ISORTERTEN, BERZ-
MUSKELZELLE AUSSERHALB DES ORGANISMUS. ©

By Montrose T. Burrows.

Miinchener medizinischen Wochenschrift, No. 27, 147351475 and 2 figures.

FISH (FUNDULUS) EGGS.
_Am. Jour. of Physiology, Vol. XXXI, 131-144 and 9 figures.

Jour. of Biological Chemistry, Vol. XI, -485-441.

PREPARATION OF MATERIAL FOR HISTOLOGY AND.EM-
BRYOLOGY, WITH AN APPENDIX ON THE ARTERIES

AND VEINS IN A 30 MM. PIG EMBRYO.

By J. F, McClendon.

Anatomical Record. Vol. VII, 51-61 and 3 ‘figures.

5. ECHINOCHROMS, A RED SUBSTANCE IN SEA UDCHINS. :
By J. F. McClendon.

. THE EFFECTS OF ALKALOIDS ON THE DEVELOPMENT OF
By J. F. McClendon.

~

AN EXPERIMENTAL STUDY OF RACIAL DEGENERATION
IN MAMMALS TREATED WITH ALCOHOL *

CHARLES R. STOCKARD, Pu.D.
NEW YORK

It is recognized, by most observers who have studied the subject, that
alcohol may play an important réle in the causation of monstrosities and
of structural defects predisposing to later disease. This view is based
largely on observations on defective human beings, and the probability
of its truth is sufficiently established to warrant further careful experi-
mental analysis.

The quality of an offspring depends on two factors, the perfection of
the germ cells from which it arises and the nature of the environment in
which it develops. Diseased and weakened germ-cells give rise to a
defective individual under all circumstances, while perfect germ-cells
produce a perfect offspring only when the embryo develops in a normal
or favorable environment. These facts may be readily demonstrated in
lower vertebrates in which the development of the egg is outside the
mother’s body. The egg or spermatozoon in such cases may easily be
chemically modified or injured before fertilization, and the embryo itself
may be affected in various ways during its development by subjecting it to
unusual surroundings, either physical or chemical. In other animals,
such as mammals, in which the embryo develops internally, the proposi-
tion likewise holds true. In these animals, however, the problem is more
difficult to completely analyze. ‘The reactions of the parental body, the
secondary conditions induced by the experimental treatment and other
sources of error should be fully considered in determining whether an
effect shown by the offspring is directly due to the applied stimulus or to
secondary conditions. In the lower vertebrates it has been shown that
given doses of certain substances induce definite developmental defects.
The defects are directly due to the treatment. Is it possible by the
addition of certain chemicals to the mammalian body to obtain similar
definite changes in either the germ-cells or the developing embryo?

In the present paper I shall endeavor to show that alcohol does act
directly on the germ-cells of mammals to a sufficient degree to render
them incapable of producing normal offspring, and further, that similar
treatment administered to the pregnant female may likewise act directly
on the developing embryo so as to modify its resulting structure.

*From the Anatomical Laboratory, Cornell University Medical College.
*Manuscript submitted for publication May 19, 1912.

First to appreciate fully the general status of the problem it is well to
consider in a somewhat critical manner the literature pertaining to the
actions of alcohol and other substances on the reproductive glands and
developing embryos of man and lower animals.

DISCUSSION OF LITERATURE

There is an abundant literature relating to the effects of alcohol on the
offspring, though little of it is scientifically reliable. I have attempted
to select those cases which seem most trustworthy. Since we are more
interested in the general problem of the effects of parental poisoning on
the germ-cells and the embryo in mammals I have also collected the works
relating to injurious substances other than alcohol. The observations and
statistics on human beings in various countries are reliable only in so far
as they may be substantiated and borne out by controlled experiments on
lower animals, Yet in the light of animal experiments many of these
human records become of surprising interest, although few if any of them
may be accepted entirely as they stand.

EFFECT ON THE MALE GERM-CELLS

It is a well known and universally accepted fact that alcohol does
cause changes and degeneration in many of the body tissues of man. The
question naturally presents itself, How, then, can the reproductive tissues
escape? Nicloux and Renault have shown that alcohol has a decided
affinity for the reproductive glands. In the testicular tissues and the
seminal fluid an amount of alcohol is soon present which almost equals
that in the blood of a person having recently taken alcohol. The pro-
portion of alcohol in the testis as compared with that in the blood was as
2 to 3, and in the ovary of female mammals as 3 to 5. The genital glands
show as great an affinity for this substance as does the nervous system.
From these observations it must necessarily follow that alcohol may act
on the ripe spermatozoon shortly before the time when it fertilizes the
egg, and since an affected spermatozoon gives rise to a defective individual
we have a probable explanation for many of the recorded defects attrib-
uted to drunkenness at the time of conception. A male, even for the
first time, in a state of acute intoxication, is probably more apt to beget
an abnormal offspring by fertilizing an egg at this particular period than
is a non-intoxicated male although a frequent user of alcohol. The experi-
mental data on the sensitiveness of the spermatozoon and the observations
on the presence of alcohol in the seminal fluid warrant this statement.

1. Most of the literature is devoted to considerations of disease and insanity
statistics and the family records of degenerates. The data are often collected in
a careless fashion so that the actwal observations are not always scientifically
correct though the records are carefully and fuily computed.

3

Lippich claims to have observed ninety-seven children resulting from
such conceptions. Only fourteen of these were without noticeable defects.
Eighty-three of them showed various abnormal conditions, twenty-eight
were scrofulous,” three had “weak lungs,” three showed different atrophic
conditions, one watery brain, four were feeble-minded, etc. Others have
made similar observations. Sullivan reported seven cases of drunkenness
during conception which are fairly authentic. Six of the offspring died in
convulsions after a few months, and the seventh was still-born.

Thus one finds proof by Nicloux and Renault that alcohol does reach
the reproductive glands and, therefore, may affect the egg or sperm-cell,
and observations seem to indicate that this effect expresses itself in the
condition of the resulting offspring. Experiments on lower animals
support the probability. When the perfectly normal spermatozoa of frogs
are treated with z-ray or radium, Bardeen and O. Hertwig have shown
that normal eggs fertilized by such spermatozoa all develop abnormally.
Todde found that the offspring from alcoholized roosters were not quite
normal and that the roosters did not succeed as well as normally in
fertilizing eggs.

Combemale, 1888, was the first to experiment on the influence of
alcohol on the mammalian offspring. He treated a dog for eight months
with absinthe (11 gr. per day per kilo of animal weight) and paired
this alcoholized dog with a normal bitch. ‘Twelve young resulted; two
were born dead, three died within fourteen days and the others died
between thirty-two and sixty-seven days of intestinal catarrh, tubercu-
-losis, ete. In a second experiment both parents were mated while normal,
then the female was made drunk for twenty-three days (2.75 to 5 gr.
absinthe of 72 per cent. per day per kilo). Of six young three were
still-born, two had normal bodies though of weak intelligence, while one
moved slowly and was very stupid. The last individual, a female, was
later paired with a normal intelligent non-alcoholic dog. She gave only
three young; one was deformed, club-footed with abnormal] teeth, the
second had a patent ductus arteriosis and died after fourteen days, while
the third was poorly muscled in the hinder parts and died a few hours
after birth. Thus the effects in the second generation are as pronounced
as in the first although neither parent had themselves received any
alcohol. The only criticism against Combemale’s experiments is that an
insufficient number of animals was used. Dogs often give defective pups
and these may have been from poor stock, though such an interpretation
is really not probable, and his results are supported by subsequent workers.

2. Imbault, F.: Contribution a l’étude de la fréquence de la tubereulose chez
les aleooliques. Thése de Paris, 1901. Imbault found that tuberculosis was about

as common among the children of alcoholic parentage as among those of tubereu-
lous parents.

1

Hodge, in 1897, obtained similar results. From one pair of alcoholic
dogs he obtained twenty-three pups, eight were deformed and nine were
born dead, while only four lived. In a control set forty-one individuals
lived, four were deformed and there were no still-births.

Laitinen treated rabbits and guinea-pigs with various doses of alcohol
and studied chiefly the changes in body conditions as to resistance against
disease toxins, etc. He has also recorded observations on the offspring
produced by these animals during the experiment. He is apparently
more interested in the problem of the misuse of alcoho] than in the
scientific study of the influence of injurious substances on the offspring
and in his enthusiasm to prove the point with extremely small doses of
alcohol he fails to fully consider both sides of his own tables.

He used daily doses of alcohol as small as 0.1 ¢.c. per kilo of animal
weight. This would amount to a small glass (200 c.c.) of beer per day
for an adult man. His tables on careful study fail to show that so little
alcohol actually does injure the offspring of the treated animals.

With alcoholized rabbits Laitinen finds that only 38.71 per cent. of
the young live, while 61.29 per cent. are still-born or die shortly after
birth. In the control, however, only 45.83 per cent. lived, while 54.17 per
cent., more than half, were still-born or died shortly after birth. The
animals were kept all together in a general cage and the pregnant females
were only separated shortly before the young reached term. This is
scarcely an approved method in breeding experiments, and the fact that
young rabbits are so delicate and are born in a rather poorly developed
state makes their careful handling necessary. The fact that more than
half of the control young die, 54.17 per cent., would indicate the danger
of drawing conclusions from a death-rate only 7 per cent. higher among
the offspring of the treated animals.

The case of the guinea-pigs is also indifferent, 78.26 per cent. of the
control young lived, while 21.75 per cent., or a little more than one-fifth,
of them died. The large majority of the young of treated parents also
lived, 63.24 per cent., while 37.76 per cent. died. In both sets more of the
young lived than died. Guinea-pigs are easily reared and are born in a
well-developed condition. On the other hand, in both of the rabbit sets
more of the young died than lived.

Results which I shall record below show that larger doses of alcohol
do produce definite effects on the offspring. My experiments have been
performed in a different manner and from another point of view. The
primary object has been to regulate or control the type of development in
mammals in a definite fashion as I had succeeded in doing with lower
vertebrates. In these experiments it will be demonstrated that an
alcoholized male guinea-pig almost invariably begets a defective offspring
even when bred to a vigorous normal female.

5

Résch was the first to study the reproductive glands of alcoholics, in
1837, and found degeneration of the testicles. Lancereaux described a
parenchymatous degeneration of the seminal canals. Simmonds (1898)
found azoospermia in 60 per cent. of cases of chronic alcoholism; 5 per
cent. of these men were sterile. Kyrle reported three cases of total
atrophy of the testicular parenchyma in which death had resulted from
cirrhosis of the liver due to alcohol. Kyrle attributed the atrophy of the
testicle to the cirrhosis of the liver and not to chronic alcoholism.

Bertholet (1909) made an extensive examination of the influence of
alcohol on the histological structure of the germ glands, more particularly
on the testicles of chronic alcoholics. He found testicular atrophy in
alcoholics with no cirrhosis of the liver. Bertholet observed partial
atrophy of the testicles in the majority of seventy-five chronic alcoholics.
These men died between the ages of 24 and 57 years, the greatest mor-
tality being between 30 and 50 years. In thirty-seven cases, excluding
syphilitics, a microscopical examination showed a more or less diffuse
atrophy of the testicular parenchyma and a sclerosis of the interstitial
connective tissue. ‘The testicles were small and hard. The canals were
greatly reduced in size and their lumina obliterated. Spermatogonia
were atrophic. It was generally impossible to differentiate spermatocytes
or spermatids. There were no dividing cells and no spermatozoa. The
thick basal membrane of the canals was formed of connective tissue
lamelle with concentrated spindle cells. These conditions with slight
variations were found in twenty-four cases. Such atrophic structures
- were already present in a drinker only 29 years old. In four cases of
cirrhosis of the liver the testicular atrophy had not progressed very far
and spermatozoa were still present. In five cases the microscopical con-
ditions were less marked.

While these appearances of the basal membrane may also be observed
in non-alcoholics, the extreme conditions of atrophy of the testicles were
only found in alcoholics. Observing the testicles of non-alcoholics that
had died of various chronic illnesses such as tuberculosis, no atrophy of
the testicles or thickening of the membrana propria was found. Two
such old men of 70 and 91 years still possessed spermatozoa in the canals.
Bertholet concludes that the atrophy he has observed cannot be due to old
age, but is due to the hurtful effects of chronic alcoholism on the repro-
ductive glands.

Bertholet has also reported an atrophy of the ovary and ova in female
alcoholics. Weichselbaum has confirmed the observations of Bertholet at
his institute in Vienna.

Bertholet’s observations are most important and his drawings bear
out his statements. On the other hand, it is certain that the chronic
alcoholic is not so often rendered sterile as his study might lead one to

6

believe. Judging from the statistics it is not rare to find alcoholics with
large families. My experiments on animals may not be of sufficient
duration at the present time, yet I have male guinea-pigs that have been
almost intoxicated on alcohol once per day for six days a week extending
over a period of nineteen months. ‘These animals are still splendid
breeders. Nineteen months of a guinea-pig’s existence is proportionally
equal to a good fraction of a human life. Many of these animals have
been killed and their testicles examined microscopically and found to be
normal. In some cases where a male had failed to succeed in impreg-
nating the female for several times, he was partially castrated, one testicle
being taken out. In this case the testicle was found to be normal and the
same male has since given offspring by other females. Ovaries have been
examined in a similar way, and in no individual has the alcohol treatment
caused a visible structural change in the reproductive glands. The actual
physiolegical proof of the efficiency of the organs is shown by the ability
of all animals to reproduce. The important point which I shall show in
the following pages is that although there is no visible structural change
in the germ-cells, nevertheless, they have been modified chemically to an
extent sufficient to cause them to give rise to defective embryos or
weakened individuals which die shortly after birth.

Schweighofer has recorded an interesting individual case. A normal
woman married a normal man and had three sound children. The
husband died and she married a drunkard and gave birth to three other
children; one of these became a drunkard, one had infantilism, while the
third was a social degenerate and drunkard. The first two of these
children contracted tuberculosis, which had never before been in the
family. The woman married a third time and by this sober husband she
again produced sound children. This is an important human experiment.
The female was first tested with a normal male and gave normal off-
spring; when mated with an alcoholic male the progeny were defective as
a result of his poisoned condition. She was again tested with a normal
male and found to be still capable of giving sound offspring. A number
of such cases are on record.

Schweighofer states from a mass of observations that the offspring of
drunkards, themselves of good sound families, show much degeneracy and
defective conditions. :

Other substances than alcohol seem to act directly on the germ-cells of
mammals. Constantine Paul long ago pointed out that the children of
people working in lead were often defective. He made the interesting
observation that when the father alone was employed in such work his
children were affected by it.

All of the above experiments and observations refer more particularly
to the action of injurious substances on the germ-cells of the male parent.

7

This is the crucial proof of an effect on the germ cells. The case of the
female is complex, since the substance may produce a germinal defect by
acting on the egg, or it may also directly affect the developing embryo
and thus act as an environmental influence on development.

THE FEMALE GERM-CELLS AND THE DEVELOPING EMBRYO

Herbst’s classical lithium experiments show the influence of salt
solutions on developing eggs. The experiments of J. Loeb on fish
embryos, those of Morgan on the frog and my experiments on fish all
show the marked influence of inorganic salts and organic compounds on

the development of the embryo. I showed that alcohol caused all known

TABLE ]1.—EFFECTS OF WORKING IN LEAD

2s &
n ! (apt t OF |
v Bm | Sow on Remarks
bon! = = 2 = = toy 8)
° o° rac =
sé )s68 |2es |=
Perla ee 59
Females showing lead | One of the living chil-
poisoning symptoms.. 4 15 13 | 2 dren died in 24 hours
Females working in type
foundry; previously
had nermal pregnan- | |
oS eee i 5 | 36 25, 7 Four died in first year
_ Female in type foundry; |
five pregnancies .... 1 5 | 5 | UL 4 ier ge Pantene tcc reer ne
Females working inter-
mittently; while there 3 34 ae 0 After being away for
; | | some time had healthy
Females with blue line | children
on gums, only sign of |
BOI? 2. 5... 6 rae | eee w<s . <<< on eee
Male alone exposed...... ? 32 12S eee S died first year, 4 sec-
| ond year, 5 third year
LOGI ane ee oS 120 | 83 | 387 | 22 died under three years
|

gross abnormalities of the brain in fish embryos and also gave all possible
abnormal conditions of the eyes. Other substances such as ether, chloro-
form, chlorbutanol (chloretone), etc., also had a peculiar affinity for the
developing central nervous system. These substances also act physiolog-
ically on the central nervous system of the adult.

Constantine Paul not only showed the injurious effects of lead on the
paternal germ-cells, but also recorded instructive data regarding the off-
spring of women working in lead. More recent observers have pointed
out the frequency of idiocy and other defects among the children of lead
workers. Adami has tabulated the findings of Constantine Paul as shown
‘in Table 1.

8

Forel states that acute alcoholic intoxication affects not only the brain,
but, as Nicloux has shown, the alcohol passes quickly to the cells of the
testicle or ovary and Bertholet’s observations confirm this. A conception
which takes place while the cells are in this poisoned state often results
in a feeble-minded or degenerate child. The facts furnished by experi-
ments on the eggs and spermatozoa of lower animals lend the strongest
support to this idea and there is no experimental evidence that can be
interpreted as opposed to Forel’s statement.

Chronic. alcoholics who consume daily certain amounts of alcohol
slowly injure their germ cells. By intensive use of alcohol these
cells may actually be killed or caused to atrophy. This, however, is the
extreme case and before reaching a state of atrophy the cells pass through
various grades of defectiveness. ‘The stages may show no anatomic
changes, but their physiologic state is indicated by the defective individ-
uals to which they give rise in development.

Bezzola found that in Switzerland, in the years 1880 to 1890, there
were 8,190 idiots. Most of the idiots were born in wine districts, and the
season for the maximum birth of such children was nine months after
the great national feasts, indicating, possibly, that idiots were conceived
during the period of heaviest drinking. Schweighofer found the same
relationship between the season for the greatest number of still-births
and the feast seasons in Austria.

Martin studied the family histories of eighty-three epileptic girls in
the Salpetriére (Paris). Sixty had alcoholic parents, while of the other
twenty-three alcoholism was doubtful or absent in their parents. The
sixty girls from alcoholic parentage had 244 sisters, of them 132, or 54.1
per cent., were dead; forty-eight, or 19.7 per cent., had had spasms
during childhood.

Studying the direct ancestry of 370 insane people, Jenny Koller (in
1895) found that there were twice as many drinkers as were found in
the direct ancestry of 370 sound people selected at random. Others
have recorded similar observations.

Karl Pearson and Miss Elderson studied statistically 3,000 school
children in England. They concluded that the children of alcoholics
were often heavier than those of sober parents, they were also less dis-
eased, had little epilepsy and tuberculosis and are actually cleverer in
school. They found, however, a greater mortality among the children of
alcoholics, especially of female drinkers, and concluded that only the
stronger children lived, and therefore, their quality was good.

These studies have been widely criticised, and are probably not based
on very thorough biological observations. They consider, in the first
place, only school children. It is not known whether the parents were
drunkards at the time of, or previous to the conception. The degenerate

4)

offspring of alcoholics could not enter school. The results would doubt-
less have been quite different if the inmates of an institution for defective
children had been studied. The great body of evidence from anatomic
studies of the reproductive glands of alcoholics, the animal experiments
and disease records are all opposed to Pearson’s conclusions.

The most valuable study that I have been able to find on the influence
of alcohol on the human offspring is that of Sullivan in 1899.

Sullivan emphasizes the point that while much effort has been made
to record alcoholism in the ancestry of degenerates, the important study
must be made on degeneracy in the descendants of well-observed alco-
holics. He studied the alcoholics among the female population of the
Liverpool prison and as far as possible chose cases of alcoholism that were
unaccompanied by disease or other degenerate factors.

Localization of alcoholic lesions in the body are not well worked out,
yet it is unquestionable that in the criminal, as in insane alcoholics, the
nervous manifestations of the intoxication occur with notable frequency,
while non-nervous disorders are rare or secondary. Of these alcoholic
females, thirty-one had had one or more attacks of alcoholic delirium,
twenty-four had occasional hallucinations, suicidal impulses; disorders
of cutaneous sensibility, and cramp in the extremities was noted in a
considerable number of cases. In these patients tissues other than the
nervous, so far as examination of the patients themselves could show,
_ were comparatively immune to the poison of alcohol, and this was also
true of their alcoholic relatives.

There were 100 women in the series Sullivan observed, and twenty
of these gave details of female relatives of drunken habits who had chil-
dren. ‘lo these 120 females were born 600 children, of whom 265, or
44.2 per cent., lived over two years; 335, or 55.8 per cent., died under
2 years or were still-born. Twenty-one of the women observed gave
records of sober relatives, sisters or daughters married to sober men.
The twenty-one drunken females had 125 children, sixty-nine, or 55.2 per
cent., died under 2 years; the twenty-eight sober females had 138 chil-
dren, and thirty-three, or 23.9 per cent., of them died under 2 years.
The death-rate of children from the drunken mothers was nearly two
and one-half times greater than that of the children of their near-blood
relatives who were non-alcoholic. The alcoholics, however, are poor
mothers and take little care of their children; this fact might possibly
account for the entire difference, though such a deduction is extremely
improbable.

The progressive births in the alcoholic family show interesting rec-
ords. In eighty cases the number of children reached or exceeded three.

The tabulation shows an increasingly poor condition. The records of
two individual cases may be mentioned by way of illustration:

10

Case 5: Three first children healthy, fourth of weak intelligence, fifth epileptic
idiot, sixth still-born, and finally an abortion.

Case 10: First child survived to adult life, second died of infection as child,
two infants then died in convulsions in first few months, then a still-birth.

These records stand in interesting contrast with those known for
syphilitic mothers in which each conception seems to be more and more
nearly successful until a weak offspring is born, and finally such a mother
may give birth to an apparently normal child. The syphilitic is grad-
ually becoming less diseased and is overcoming the toxic condition as
time goes on, while these alcoholic women are on the contrary becoming
more and more saturated with the poison, and for this reason each suc-
ceeding birth is more decidedly defective.

TABLE 2.—SHOWING PERCENTAGE CF STILL-BORN AND CHILDREN WHO DIED IN
AN ALCOHOLIC FAMILY

Died or 5
Cases Still-Born, Soe
Per cent. ;
Hainstabornneceiynaceoc neers 80 SBhl | 6.2
SEONG Warrin Soobsosooease 80 | 50.0 11.2
Minds bonne seie et eretenagt 80 52.6 7.6
Fourth and fifth born ..... Wity! 65.7 10.8
Sixth to tenth born.....:. 93 72.0 Niiee!

The records were worse for women who had begun drinking some
time previous to the first conception. In thirty-one cases they had been
drinking for at least two years before the first pregnancy. Of 118 chil-
dren born to these, seventy-four were still-born or died in infancy, giving
62.7 per cent. as compared with a death-rate of 54.1 per cent. for the
others of the series.

Tn only thirty-nine of the cases were the women’s parents sober peo-
ple, yet the records of the offspring from these women were equally as
bad as those from the sixty-one mothers who had alcoholic parents. This
is a significant fact, since it indicates most strongly that the defective
children are due to the direct effect of alcoholism and not to other degen-
erate conditions. Sullivan recorded seven known cases of conception dur-
ing a state of drunkenness; six of the children died in convulsions in a
few months, while the seventh was still-born.

Another observation by Sullivan which indicates that the alcohol
as such is the cause of defectiveness was the fact that mothers impris-
oned during pregnancy gave birth to a better child since the drinking -
was stopped.

Ala

Sixty per cent. of the children of all these mothers died in convul-
sions. This is a common manner of death for the offspring from the
alcoholic mammals I have studied.

Kende found that of twenty-one families in which the father and
mother both drank, ten were childless, while of the twenty-four children
in the other eleven families, sixteen died early and only three were
entirely normal. In eighteen families in which only the father drank,
but three children in twenty-one were entirely sound, while there were
many abortions and several cases of sterility.

There are numerous statistical facts showing a large percentage of
alcoholics in the ancestry of prostitutes, degenerates and other inferior
classes. Ail of the studies seem to show that alcoholism and the degener-
ate condition tend to occur in the same family, and Sullivan seems to
control the case by showing that in some instances, at least, alcohol is
the cause of degeneracy.

The real, crucial proof of the direct action of alcohol must come,
however, from experiments on lower animals, where the sources of error

ay be entirely controilled.

Adami states: “The general belief (and we regard it as well founded)
is that the children of the sot are as a body of lowered intelligence and
vitality with unstable self-control.” He recognizes the great difficulty
of statistically proving this in man, since alcoholism is so often the
accompaniment of weakness and hereditary taint, and may not be the
primary cause of the condition in many families. With animals, how-
ever, the experimenter is enabled to prove that alcohol does induce a pri-
marily degenerate condition.

One could continue to enumerate records showing the effect of alco-
holism on the human offspring, yet a sufficient number of studies have
been considered to show how strongly indicative the evidence is that
alcohol is really the direct cause of defects in many cases. There is
also little doubt that alcoholism is sometimes acquired by perfectly nor-
mal human beings, and when the tissues of such people become affected
by alcohol they no doubt give rise to defective and abnormal offspring.

It is, however, an undeniable fact that alcoholism in man is very
frequently an accompaniment of various degenerate conditions, and
these conditions are oftentimes within themselves sufficient to account
for further degeneration in the offspring. We shall, therefore, consider
more fuliy at this point the evidence furnished by animal experi-
mentation.

ANIMAL EXPERIMENTS

As stated above, the problem is broader than the subject of alcohol-
ism. If it is shown that any toxic substance can act on the germ cells
or developing embryo in such a manner as to change or modify its

12

development, it necessarily follows that alcohol may induce a more or
less equivalent condition, since it is definitely known to act on all animal
tissues. I shall, therefore, mention the experiments with alcohol in par-
ticular, and.at the same time consider other of the striking examples
of environmental effects on the developing eggs of lower animals.

H. E. Ziegler treated sea-urchin’s eggs with ethyl-alcohol. A 1 per
cent. solution in sea-water delayed development, a 2 per cent. solution
also delayed development and caused abnormal embryos, while a 4 per
cent. solution prevented all development. The peculiarly typical larvee
of the sea-urchin which Herbst induced by the addition of lithium salts
to sea-water have been mentioned above. Herbst’s experiments furnish
a striking example of a characteristic response on the part of the devel-
oping organism to a definite chemical treatment. Morgan obtained
similarly definite results by treating frog’s eggs with lithium, and I
have shown a somewhat comparable response for the fish’s egg. Other
salts may give the same types of larve, as occurred in these cases, as
McClendon has shown for the fish, and as I previously pointed out in
several of my studies on the cyclopean defect. Yet with certain doses
of given substances one gets greater numbers of the same defect than
with any other treatment. It is not surprising that a few individuals
of any one deformed type may occur in a number of different solutions.
The important fact is, that with a particular treatment one is able to
obtain on all occasions a large number of embryos exhibiting a perfectly
clear-cut, definite defect.

Ridge got decided results by treating the eggs of the blue bottle-fly
and frogs with alcohol. In solutions of 1/100 per cent. alcohol in
water the development was slow. In 1/20 per cent. solutions develop-
ment proceeded for only a.short time and the eggs died. In one per
cent. alcohol only one or two eggs started.

Ovize made an interesting observation on the influence of alcoholic
fumes on developing hen’s eggs. An incubator containing 160 eggs was
in a cellar in which wine and brandy were being distilled. Seventy-
eight chickens hatched; of these twenty-five were deformed and forty
died during the first three or four days. Of the number unhatched,
one-third were deformed, and 3 to 4 per cent. had only developed a
short way.®

Féré has experimented extensively with the influence of alcohél on
the developing hen’s egg. Alcohol was injected into the albumen in
some experiments, while in others the eggs were placed under bell jars
and exposed to the fumes of evaporating alcohol. Enough of the fumes
penetrated the shell and entered the egg to affect the subsequent develop-
ment of the embryos. When eggs were placed in the incubator after such

3. These results by Ovize were taken from Forel’s review.

15

treatment they developed more slowly than the control and a large
umber of malformed embryos resulted. The abnormalities were vari-
able, yet many had defective nervous systems and a number of the
embryos exhibited eye defects. Féré made no attempt to analyze the
cause of the different types of deformities, and in fact he paid little
attention to the structure of the defects. Yet he showed most decidedly
that alcohol fumes do affect the developing embryo, as one might have
inferred from the preceding observations made by Ovize.

I have repeated Féré’s experiments at some length during the past
two years and can confirm his results. My object has been to regulate
the treatment in such a manner as to get definite types of defects with
certain intensities of treatment. Up to the present time I have only
partially succeeded in doing this, though in several experiments the
delay in development and the general type of the defects has been rather
constant. This treatment of hen’s eggs with alcoholic fumes is one of the
most convincing and easily performed demonstrations of the influence
of alcoho! on development.

Féré also experimented with hen’s eggs to show the influence of dif-
ferences in temperature during incubation and many other physical and
chemical factors. Ali unusual conditions affected the development of
the embryo. Feré also developed hen’s eggs in glass dishes after remoy-
ing them from the shell. Preyer and Loisel had previously done similar
experiments, but they carried the embryo for only a day or so, while
Féré succeeded in keeping the egg developing for six days. Some of
these embryos develop abnormally.

I have recorded a number of experiments on fishes’ eggs which show’
the decided effects of alcohol and a large series of other substances on
embryonic development. Alcohol and various anesthetics showed a
peculiar affinity for the developing nervous system and organs of special
sense. In many cases other organs and parts of the embryos were appar-
ently normal. Many of the deformed individuals hatched and lived for
some time, swimming about and feeding in a typical fashion.

With alcohol solutions of given strength definite defects were induced.
In some experiments dozens of embryos with typical brain and eye
defects occurred, while few or no other types of deformities existed.
The experimenter has the power in these cases to predict with at least
a limited degree of certainty the type of deformity which will result from
a definite intensity of a particular treatment. Embryonic development
in such cases may really be regulated or controlled.

We have already considered a number of experiments on mammals
which show that alcohol and other injurious substances affect the qual-
ity of the offspring. Jn treating mammals the case is not so simple as
in treating the eggs of lower vertebrates which develop outside the

14

parent’s body. The effects in mammals may not be due directly to the
substance used, but rather indirectly to the changed conditions the sub-
stances have induced in the body of the parent. It is important for this
reason to know whether certain substances come in direct contact with
the germ cells of the individual. As before mentioned, Nicloux and
Renault have shown that alcohol may be readily found in the seminal
fluid of a man shortly after drinking it. Thus the spermatozoa may
come to float or swim in a weak solution of alcohol.

In the case of female mammals, Nicloux has carefully demonstrated
the passage of alcohol from the blood of the mother into the tissues of
the embryo. The following tabulation readily shows the results of his
experiments on dogs and guinea-pigs:

TABLE 3.—PASSAGE OF ALCOHOL FROM Tie MOTHER TO THE FETUS

| oc
Vee cen lee
liner Se ee = = = °
2.5 || 2 S = =) =)
ay ss 53 z PG 2
eae. Lv 2 ae. a 7 Sia ue es
| Se ies el 50% See Sie oa8
oe = pee na + sy i) — 0) —= 49
3) <a <i am <a <p @r es
O43 & Oagn sy ees =. =, D
|q5 9 8 |) OS S59) SSeS ees ome eesenters ae!
| 9S de |) poe = hes aS a os & =e
Ges |.8 6,0 | Sco) eterced esees Sie
Inq (ote | asf ye <r <= t0o=
1. Guinea-pig 5 5/6 0.56 0.31
2. Guinea-pig 5 1 0.47 0.35 Reete ‘6 nbs od
3. Guinea-pig 2 ] 0.20 Aree 0.10 0.12
4. Guinea-pig 1 ] 0.13 oie 0.081 0.086
5. Guinea-pig | 0.5 1% 0204055 eae 9.015 0.02
6. Dog 3 | es Osi | O37) Ons | U2

After a short period of time the amount of alcohol in the blood of
the fetus is about equal to that in the blood of the mother, while there
is really more alcohol in a given weight of the tissues of the fetus than
is to be found in an equal weight of liver tissues from the mother.

The reality of the passage of alcohol from the mother to the fetus
demonstrates the possibility of the intoxication of the fetus. Therefore,
nervous disorders, anesthesia, etc., of the late fetus may result as a
consequence of alcohol in the blood, while the developing embryo or early
fetus will show the effects by an abnormal formation of the nervous
system.

Thus the results of the experiments of Mairet, Combemale and Hodge
on dogs are readily explained as the direct influence of alcohol on the
paternal germ cells in the case of the treated male, or on the developing
fetus within the body of the alcoholic mother. The great number of
human records briefly referred to above are also readily interpreted as

the result of direct alcoholic action on the germ cells and the developing
embryo.

15

The experiments on mammals do not then really differ greatly from
those on the lower vertebrates where the externally developing eggs are
placed directly in various unusual solutions, since the egg or embryo
although within the mother’s body is readily bathed or impregnated by
the alcohol contained within the mother’s blood.

The only experiments with alcohol on lower mammals which do not
fall completely in line with the above records are those recently recorded
by Nice. He has fed mice on alcohol. Each day 2 c.c. of 35 per cent.
alcohol was added to crackers and milk and placed as food for each
mouse. Instead of drinking water the mice could drink 35 per cent.
alcohol from a syphon which prevented evaporation. Animals treated
in this way gained in weight over the control. The offspring from these
alcoholic mice excelled all the other mice in growth, even when they
themselves were fed alcohol. The young grew faster, however, when not
given alcohol. Nice treated other mice with tobacco fumes, nicotin and
eaffein. The fecundity of the alcohol, nicotin and caffein mice was
greater than the control while those treated with tobacco fumes had
almost twice as many young as the control. The mortality of the off-
spring from the treated mice was, however, greater than from the control.
- None of the control young died, while 17.3 per cent. of the nicotin
young and 11.1 per cent. of the alcoholic young died soon after birth.
There was only one abortion, no still-births and none of the young were
deformed.

Mice may possibly be peculiarly resistant to these drugs, though I
should rather think that in the case of alcohol, at least, the animals
received too little to give a pronounced effect, though it was sufficient |
to cause a certain fatality among the young. Weak alcohol mixed with
erackers and milk no doubt rapidly evaporates. The animals possibly
waited until a certain amount of the alcohol had disappeared before they
ate their food, and, of course, the amount of alcohol they took instead
of drinking water was very small. Mice may easily be kept on a cracker
and milk diet without ever receiving water. One cannot deny, however,
that the mice did receive enough alcohol to cause them to fatten more rap-
idly than the control, and probably to cause the death of some of their
offspring.

Carriére has shown that when guinea-pigs are inoculated with various
soluble products of the tubercle bacillus for several months that the
number of offspring is diminished. He sometimes observed the death of
the fetus or premature death of the young, while many of the living
young had feeble constitutions. The action was produced when either
parent was impregnated with the poison.

Mating together two inoculated animals gave 52 per cent still-born
young; 28 per cent. of the living young died under sixteen days and

16

only 20 per cent. of the young survived. When the female alone was
inoculated 26.9 per cent. of the offspring were still-born, 34.6 per cent.
died under sixteen days and 38.4 per cent. of the young survived. The
matings with the male alone inoculated gave 16.6 per cent. still-born,
10 per cent. dying under sixteen days and 73 per cent. of the offspring
survived. Thus the effect of the toxin is shown on the germ cells of both
sexes,

Lustig’s experiments of inoculating fowls with abrin gave results
parallel to those recorded by Carriére. The. offspring were less resistant
to inoculations of abrin, just as the guinea-pigs were to the tuberculosis
extracts when compared with control animals of the same age.

Mall has clearly shown in his monograph on the causes of human
monstrosities that poor nutrition and abnormal environment are most
potent factors. Only 7 per cent. of the uterine pregnancies examined
gave monsters, while 96 per cent. of the tubal pregnancies produced
abnormal embryos.

Ballantyne has presented in his “Antenatal Pathology” a most com-
prehensive consideration of the part played by abnormal environment
and disease in the causation of monstrosities and developmental defects
in general.

The effects of malnutrition or poor environment on the developing
embryo is splendidly illustrated by the case of monochorial twins when
one becomes more vigorous and pumps blood from the other through the
anastomoses between their placental or umbilical vessels. In such cases
one of the twins may fail to develop certain parts and may actually lack
a heart, the heart of the superior embryo pumping blood through both
the bodies. The various degrees of the degenerate or parasitic twin is
thus produced. One individual falls behind in development and may
finally actually be included within the body of the more vigorous twin.
Double monsters may occur in which one individual is almost perfect,
while the smaller monster is attached to some part of its body.

I have given this somewhat extensive survey of the literature in order
to show that an abundance of evidence exists at the present time to
indicate that the course of embryonic development may be readily modi-
fied. It is also clearly shown that the germ cells of various animals may
be directly affected by different chemical treatments to such a degree
that they give rise to defective individuals. ‘The experiments of O.
Tertwig and Morgan on the chemical production of spina bifida in large
numbers of tadpoles and my experiments on the constant production of
typical cyclopian monsters by subjecting developing fish eggs to definite
chemical treatments strongly indicate that the manner of embryonic
development may be definitely regulated. This exact regulation or con-
trol of development is the important goal of experimental teratology.

AG

The problem is now in its beginning, since the actual influence of various
treatments is known to be expressed in the resulting type of embryonic
development.

The studies on alcoholism in mammals have failed to produce any
convincing evidence of the specific actions of this poison. Yet the sta-
tistical studies on defective human beings would indicate that alcohol
had a special affinity for the developing nervous system. My experiments
on the influence of alcohol on the developing fish embryo demonstrated
that alcohol did have a specific affinity for the central nervous system,
and caused the brains of these embryos to exhibit numerous deformities,
while the organs of special sense were also affected.

METHOD AND RESULT

The experiments here recorded have been undertaken in order to
ascertain whether alcohol did exert a marked influence on the germ cells
and developing embryos of mammals, and, if possible to demonstrate the
nature and mode of action of this influence. I have used alcohol as an
agent, since it may be given to guinea-pigs without greatly disturbing
their normal physiological processes, and so does not produce marked
conditions which might secondarily affect the results. Alcohol may
remain as such in the blood and tissues of a mammal, and so may act
directly just as it would when added to the sea-water in which fishes”
eggs were developing. I have studied its effects experimentally on the
eggs of lower vertebrates and am familiar with the defects it produces in
these animals. It is an active substance and, therefore, for these many
reasons lends itself admirably to experimental use.

The experiments have been conducted on guinea-pigs, since they
breed fairly rapidly and rear their young without much difficulty in the
laboratory. Strong healthy stock has been chosen and the animals have
been carefully handled. All have remained in vigorous health and most
of them have increased in size and fattened during the progress of the
experiment. The males and females have been kept carefully separated
and individual pairs mated from time to time.

The animals are first tested by normal matings and found to produce
normal offspring. The alcoholic treatment is then begun on a given
number of individuals and males and females mated in different combi-
nations according to whether they are alcoholics or normal. An alco-
holic male is mated with a normal female, the paternal test. This is the
erucial test for influence on the germ cells, as here the defective offspring
must be due to the chemically modified spermatozoon from which it arose,
since the egg, and the mother in which the embryo developed, were both
normal.

18

Normal untreated males are paired with alcoholic females, the mater-
nal test. Here the defective offspring may be due either to a modified
ovum or to the fact that it developed in a mother with alcoholic blood,
therefore supplying an unfavorable developmental environment. Lastly,
its condition may be due to both of these causes. ‘The mammalian mother
has two chances to injure an offspring, either by producing a defective
egg, or secondly by supplying an unfavorable or diseased environment
in which the embryo must develop.

The final combination is the mating of alcoholic individuals. This,
of course, offers the greatest chance for defective offspring.

Alcohol is administered to the guinea-pigs by inhalation. At first
it was given with the food, but the animals did not relish it, and there-
fore took less food. It was then given by stomach tube, but this method

Fig. 1—Tank for alechol treatment. Animals are placed on the wire screen
in the closed tank and inhale the fumes of aleohol evaporating from the cotton
below the screen.

so upset the animals that the results might have been modified by their
poor bodily condition and the bad state of their stomachs. The inhala-
tion method is entirely satisfactory, the guinea-pigs thrive and usually
gain in weight during the experiment, they have good appetites and are
in all respects apparently normal. The only indication of the effects of
the treatment is shown by the quality of offspring they produce.

The apparatus used for giving the alcohol consists of an air-tight
copper tank 36 inches long by 18 inches wide and 12 inches deep, with
a sloping bottom draining to the center. Over this bottom is placed a
wire screen and below the screen cotton soaked with 95 per cent. alcohol
is spread (Fig. 1). The tank is closed and allowed to stand until the

19

atmosphere within is saturated with alcoholic fumes. A ventilation
system is so arranged that a given quantity of alcohol fumes may be
driven through the tank in a given time, but it has not seemed advisable
to use this device, as the degree of intoxication is a better index to the
physiological respense of the animals, since their resistance to a given
amount of the fumes is changeable. The guinea-pigs, three or four at
a time, are placed on the wire screen above the evaporating alcohol, the
tank is again closed and the animals are allowed to remain until they
begin to show signs of intoxication, though they are never completely
intoxicated. They usually inhale the fumes for about an hour. The
animals are treated in this way for six days per week and some have now
been treated over a period of about nineteen months. None of the effects

Fig. 2.—Breeding cage with fume tank attachment. Pregnant females are
kept in this cage and may be driven through the drop door into the fume tank.
Handling pregnant animals during the treatment is thus avoided.

are due to want of air, since the same number of guinea-pigs may remain
for hours in this closed tank without showing any signs of discomfort
when there are no fumes present.

In order to avoid handling the females during late pregnancy, a
special treating cage is devised for them. An ordinary box run with a
covered nest in which the animal lives is connected by a drop door with
a metal-lined tank having a similar screen arrangement to that described
for the general treatment tank (Fig. 2). The pregnant animal may be
driven daily into the tank and thus treated with alcohol fumes through-
out her pregnancy without having to be handled or moved about in any
way that would tend to disturb the developing fetus.

During the vapor treatment the animals usually react in a manner
quite similar to their behavior in weak fumes of ether or chloroform.

20

The majority of them sit quite motionless and sniff their noses for a
time and then become somewhat drowsy. A few individuals, however, are
excited by the treatment and run about the tank, becoming sexually
excited, and many often fight other animals savagely. One of the
males fights and bites so vigorously while taking the fumes that he has
to be treated separately from all others. The fumes then have a differ-
ent influence on the behavior of different individuals in much the same
way that alcoholic intoxication expresses itself differently on different
human beings.

During the first few weeks of the treatment the fumes cause the
eyes to water so that tears run over the face. The nose and mouth also
become moist and the animals sniff almost constantly. ‘The fumes are
very irritating to the mucous membranes at first. The cornea
becomes irritated and finally opaque in some instances, so that the eye
takes on a white appearance. The tissues seem, however, to develop a
resistance to the fumes. The eyes become clear after a few months and
never again become opaque. ‘lhe nasal mucosa also ceases to secrete
excessively unless the animal is left in for an unusually long time.

Many of the guinea-pigs have been killed after treatments of differ-
ent duration up to fifteen months, and all of their viscera carefully exam-
ined. In no case have I found any changed structures due to the alcoholic
treatment. The lungs, liver, stomach, intestines, kidneys, reproductive
glands, brain and all other parts appear perfectly normal. The general
health and behavior of the animals also indicate that they are in good con-
dition. As before mentioned, several animals have been partially castrated
during the experiment. One of the reproductive glands was removed and
examined microscopically. In all cases the germ cells, ova or spermatozoa,
as the case may be, were found to exhibit perfectly normal structure. One
cannot claim, therefore, that this treatment is excessively severe or greater
in proportional amount than the alcohol a human being often takes. The
fact is that these animals have never been completely intoxicated, but
receive only enough aleohol six times per week to affect their nervous
states. They may be compared to a toper who drinks daily but never
becomes really drunk.

While the bodies of these animals display no direct effects of the
alcohol, the conditions of the offspring to which they give rise show most
strikingly the effects of the alcoholic treatment. The results of mating
the alcoholized guinea-pigs are summarized in Table 4.

Fifty-five matings of treated animals have been made. Forty-two of
these have now reached full term and are recorded. Thirteen matings
are not yet due. From the forty-two matings only seven young survived,
and six of these are stiil living, five of which are runts, though their
parents were unusually large, strong animals (Figs. 4 and 5).

The conditions of the animals in the mating pairs are shown in the
first column of the table and the total results of the matings are indi-
eated in the following columns. The first horizontal line gives the rec-
ords when alcoholic males are paired with normal females. Twenty-four
such matings were made. Fourteen of these gave negative results, or
resulted in early abortions. Many embryos were aborted during very
young stages, and some of these were deformed, though they were gen-
erally in such poor condition after being cast out into the cages that
little could be learned from them. They were partially or completely
eaten by the mother in most cases. The males were always kept for a
number of days with the females during favorable periods, and con-
ception should have oceurred in all cases, as it did in the control matings.

TABLE 4.—EFFECTS OF ALCOHOL ON OFFSPRING OF GUINEA-PIGS

pn = Z
sales 2 =
s = a =
AS oo H | 6 = z= bs
ac i 2S io) ~ = =a
Condition of Animal 3 —— aS = Boe s
_ Haat = — = NA oO
a = Ld — _ —_-_— =
Fe Bp. a |S 2 oe mae 7S
> Paral) ear [os SI = =
} os 7) oy Pf | A eid -
Zz q Rm 1G el om L
Alcoholic male by nor- |
MA HEMET), 2.335 oe 24 14 5 8 5 | 7 5*
Normal male by alco-
holigsfemailes &....2.:.-.'. 4 ee Oey eo 3 | 3 (a) 2
Alcoholic male by alco-
holigeiemale. 9 =.=... 14 10 3 6 ts} 1 (6) 0
SUTIN Ai aieysee so eis n+ 2 42 25 S) oie 9 11 Tz
Normal male by normal |
female—Control...... 9S 0 0 0 9 | 0 | ae

*Four survivors in one litter. and one was a member of a litter of three, the
other two died immediately after birth. (a) Premature. (6) Sixth day.

7One lived to become pregnant with two young in uéero, one deformed, Fig. 3.
Other survivor normal, the mother was not treated until after first two or three
weeks of pregnancy.

Of thirty-two young born oniy seven have survived.

§One other non-alcoholic mating was made from which two young resulted;
they died after the second and fourth days, respectively, and the mother died two
days later; her diseased condition no doubt affected the suckling young. They
have for this reason not been included in the normal control.

Only ten of the twenty-four matings resulted in conceptions which ran
the full term. Half of these, or five, were still-born litters. There were
three still-born litters of two young each and two of one individual each.
Most of these were slightly premature, their eyes being closed and the
hair sparse on the bodies. (A normal guinea-pig at birth is well covered
with a hairy coat, its eyes are open and it very quickly begins to run
about actively.)

Five litters of living young were born. One litter consisted of only
one young. a weak individual that grew very little and died after six
weeks. ‘Two hitters contaimed two young each. The members of one
of these litters died during the first and fourth weeks, having been weak
and small since birth. Both of those in the other litter were in a sim-
ilarly feeble condition and died before the first month. One litter con-
tained three young; two of these died immediately after birth; the other
one is still alive, though small for its age. The fifth litter contained
four young, all of which are runts, though their parents were unusually
large animals (Figs.4 and 5). Thus out of twenty-four full-term young,
of which only twelve were born alive, but five individuals have survived,
and these are unusually small and very shy and excitable animals.

Tt is a point of some interest that all of the young animals that died
showed various nervous disturbances, having epileptic-like seizures, and
in every case died in a state of convulsion. This is commonly the fate
of feeble and nervously defective children.

The important fact in the above case is that only the father was
alcohohe, the mother being a normally vigorous animal. This experi-
ment clearly demonstrates that the paternal germ cells may be modified
by chemical treatment to such a degree that the male will beget abnor-
mal offspring even though he mate with a vigorous female. A reconsid-
eration of the figures in the first line of the table shows really how decid-
edly the injured spermatozoon expresses itself in the fate of the egg with
which it combines.

The second line of the table shows the results of matings between
alcoholized females and normal males. These matings might be expected
to give more marked results than the pervious ones, since in the treated
females not only the germ cells may be affected, but the developing
embryo itself may be injured by the presence of alcohol in the blood of
the mother. Nicloux has shown that alcohol may pass directly from
maternal blood into the embryonic tissues of a guinea-pig. The spermat-
ozoon, however, is probably a more sensitive structure than the egg and
is easily injured or killed by slightly abnormal conditions. It might
possibly be that when such a specialized cell swam for even a short period
of time in seminal fluid containing a trace of alcohol its chemical nature
would be so decidedly disturbed as to render it incapable of inducing nor-
mal development after impregnating the egg. At any rate the few cases
at present available seem to indicate that the effect on the offspring is
equally as great when it is produced by an alcoholic father as by an
alcoholic mother.

There are only four matings between alcoholized females and normal
males. One of these gave a negative result or was possibiy aborted very
early. ‘Ihree living litters were born. One of these consisted of three

rae

premature young, which died shortly after birth. The remaining two
litters each contained only one young, but these two animals survived.
One of these guinea-pigs was born after the mother had been treated for
three and one-half months. The offspring was weak and small for several
months after birth, but finally recovered and developed into a normal
animal. This guinea-pig was mated with an alcoholic male and became

pregnant. Unfortunately, she was killed by accident, and on examina-
tion her uterus was found to contain two embryos, 33 and 32 mm. in

Ai y

length. One of these embryos was deformed and showed very decided]

Fig. 3—Two embryos 32 and 33 mm. in length, taken from a female that

an alcoholic mother and was mated with alcoholic male. The upper fetus has
deformed hind legs anda poorly developed posterio1 part of the bodv: lower fetus

is normal.

degenerate and feebly developed hind legs. The posterior end of its
was also poorly formed. ‘This condition is readily seen in F
photograph of the two embryos. The abnormal one has small hind legs,
and one of them is badly folded under its body. This is of interest, sinc
all of the affected offspring of alcoholic guinea-pigs are weak in thi

hind extremities and drag their legs. Yet none were so modified as

show a noticeable structural defect except this embryo, which had on

alcoholic grandmother and an alcoholic father.

a4

‘The only other survivor from an alcoholic mother is strong and full
grown for its age. ‘The mother had been treated for only two and one-
half months when the offspring was born, so that she was normal during
the first two or three weeks of pregnancy. No doubt the early stages
of development are more easily modified to produce significant defects
than are the later. This question is being more fully tested on guinea-
pigs with experiments now in progress. I have shown, however, in

Fig. 4.—A. The animal on the left is a runt from a large alcoholic male and
a large normal female; weighs 134 gm. The animal on the right, from normal
parents, is larger although 1 month younger and weighs 147 gm.

B. The guinea-pig on the left is a runt, weighing 132 gm. from an alcoholic
father; on the right a normal guinea-pig twice as large though only 10 days
older, weighs 221 om.

treating fish eggs that the period at which the treatment is applied is
a most Important factor in determining the type of defect or modification
which will result. Certain salts, different strengths of magnesium

chlorid, for example, which give pronounced effects when added to the

«c
Or

sea-water containing eggs in early developmental stages, may really be
ineffective after the eggs have developed beyond these stages. , In the
case under consideration the offspring might. not have fared so well if
the alcoholic treatment had been started on the mother a few weeks
before conception, instead of three weeks after her pregnancy had begun.
This with other points shal! be more completely analyzed in future com-
munications on these experiments.

Fig. 5—A. Two guinea-pigs from alcoholic fathers, the left one 1 month and
10 days younger than the runt on the right.

B. The left animal is the same as above, the right another of the same
runt litter.

The four matings of alcoholic females and normal males resulted in
three living litters in all of five individuals. Three of the young were
premature and died shortly after birth, while two young survived.

26

Finally, we may consider the results of pairing two alcoholized indi-
viduals. The third line of the table summarizes these results. As might
have been anticipated, this type of mating has given the highest fatality
of all. :

Ten out of a total of fourteen matings have given no offspring or
early abortions, which were in many cases eaten by the mother. Three
still-born litters have been produced, each consisting of two young.
Only one living litter was born from the fourteen matings in which both
parents were alcoholic, and this litter consisted of but one weak indiwid-
wai which died in convulsions on the sixth day after birth. This is
indeed a decided effect of alcohol on the offspring when one compares
it with nine control matings, all of which gave living litters containing
a total of seventeen individuals, all surviving.

Two other young were produced by non-alcoholic parents and died on
the second and fourth days after birth. They have not been included
in the control since the mother died two days later in a diseased condi-
tion. No doubt the poor state of the mother had much to do with the
fate of the suckling young. She was an animal that had only been in
the experiment for a short time, and is one of the very few that have
contracted disease or died during the nineteen months of the work. This
might possibly go to show the influence of a diseased mother on the
offspring.

The fourth line of Table 1 gives a summary of the experiments.
There have been forty-two full-term matings, twenty-five of which gave
no results or early abortions; eight still-born litters have occurred, con-
sisting of fourteen individuals; only nine living litters have been born,
21 per cent. of the matings. These contained eighteen young, and but
seven of this number have survived and five of these survivors are
unusually small (Figs. 4, 5).

The bottom line of the table shows nine control matings. All have
given living litters containing a total of seventeen young, all of them
surviving. The two young that died, as stated above, were from a dying
mother and not included in the control.

Records of the successive matings of ten of the female guinea-pigs
are shown in T'able 5. The varying ways in which the same individual
has responded in different matings is noticeable. Number 10, an alco-
holie female, first mated with an alcoholic male, gave one young which
(ied on the sixth day after birth. On being remated with the same male,
No. 10, gave no result. When mated with another alcoholic male, gave
ho result. She mated again after several months with the first male and
on being killed was found to contain one embryo in utero about 2
weeks old.

Female 15, a normal guinea-pig, shows an instructive record. She
Was mated with an alcoholic male and gave birth to two still-born young.

ae

When mated with another alcoholic male she gave a negative result.
Remated with the second male she gave two young, both of which died
of convulsions within four weeks after birth. She was then mated with
a normal male as a control and gave one vigorous normal offspring
which survived.

TABLE 5.—RESULTS OF SUCCESSIVE MaTINGS OF TEN FEMALES

Animal First Mating |Second Mating | Third Mating Fourth Mating
|
No. 10 Ale. Ale. male 4,1 | Ale. male 4 | Ale. male 6 Ale. male 4,
young died 0 0 1 embryo in
in 6 days utero 2 weeks
: after
No. 12 Ale. Ale. male 5 Ale. male 5 iN Wen, TerPel eit: Ea | ier, este ate ed
0 0 0
No. 11 Ale. Ale. male 6 Ale. male 6 Ale. male 5; Ale. male 4
0 0 2 premat. 0
still-born
No. 13 Nor. Ale. male 5 | Ale. male 5 | Ale. male 4 | ..........
| 1. still-born 0 0
iIN@ lly, Nor. Etherized IDeA LS |. Ba Re ae |r oe ee
male 1 male 1
| 0 0
No. 18 Nor. : Ale. male 5 | Ale. male 5 Ales ama ein Milas ese bn
0 0 0
INO} i Nor | Etherized Etherized Miherizedey > |) es 5 ee
male 2; male 2 male 2
| 2 premat. | 0 0
still-born
No. 14 Nor. Etherized thenizeds Lilt Von e all) eich eee ce
male 3 male 3
0 0
INO; 19) Wor: Ale. male 4 Ale. male 6; Ale. male 6 | Ale. male 5:
0 1 still-born | 9 4 small, ac-
tive, only
one-half size,
but living
No. 15 Nor. Ale. male 6; Ale. male 5 Ale. male 5; Nor. male; 1
2 still-born 0 2 died fourth normal vigor-
week of con- ous young
vulsions

The other records are easily understood.

These experiments have suggested many questions still to be solved,
some of which are now being tested, such as the length of time necessary
to treat an animal before the resulting offspring is affected, whether this
time is equally Jong for both sexes, and what amount of individual varia-
tion may exist. An important point to ascertain is whether the effects
of the alcohol treatment are permanent, or does the animal recover after
a time and again become capable of giving normal offspring. One of the
most valuable problems is to regulate the treatment in such a manner
as to induce a definite type of defect with a given kind or degree of
treatment. The structure or morphology of the monsters and defective

28

offspring which occur is to be carefully studied. Many other points
might readily be suggested.

Definite and well-controlled experiments with alcohol and other sub-
stances on the mammalian offspring have not been sufficiently studied.
The work is really in its beginning, and while there is much evidence to
show that various toxic agents do affect and modify the offspring, facts
are badly needed to demonstrate the regularity and manner of this
modification. The present experiments seem to me to prove in a con-
Vincing way that alcohol may readily affect the offspring through either
parent, and that this effect is almost fatal to the existence of the off-
spring when the parents have been treated with even fairly large doses
of alcohol. Many of the cases seem to indicate further, that the tissues
of the nervous system of the offspring are particularly sensitive in their
responses to the induced conditions.

My assistant, Miss Craig, has aided me greatly throughout almost the entire

progress of these experiments. Last year during my absence abroad she assumed
entire control of the animals, and I am indebted vo her for this efficient assistance.

SUMMARY

Guinea-pigs have been treated with alcohol in order to test the influ-
ence of such treatment on their offspring. Male and female animals
are given alcohol by an inhalation method until they begin to show
signs of intoxication, though they are never completely intoxicated. They
are treated for about an hour at the time, six days per week. The
treatment in some of the cases has now extended over a period of nine-
teen months. ‘The animals may be said to be in a state of chronic
alcoholism.

Fifty-five matings of the alcoholized animals have been made, forty-
two of which have reached full term and are recorded.

From these forty-two matings only seven young animals have sur-
vived, and five of them are unusually small, though their parents were
large, vigorous guinea-pigs. The following combinations were made:

1. Alcoholic males were mated to normal females. This is the paternal
test, and is the really crucial proof of the influence of alcohol on the
germ cells, since the defective offspring in this case must be due to the
modified spermatozoa, or male germ cells, from which they arise.
Twenty-four matings of this type were made, fourteen of which gave no
result or very early abortions; five still-born litters were produced, con-
sisting of eight individuals in all, and five living litters containing twelve
young. Seven of these twelve died soon after birth, and only five have
survived. Four of the survivors are from one litter and the fifth is the
only living member of a litter of three.

2. Normal males were mated with alcoholic females. This is the
maternal test. In such cases the aleohol may affect the offspring in two
ways-—by modifying the germ cells of the mother or acting directly on

29

the developing embryo in utero. Only four such matings were tried.
One gave no offspring; three living litters were born, one consisting of
three premature young that died at birth, while the other twé litters
consisted each of one young, which have survived. The alcoholic treat-
ment in one of the last cases was only begun after the mother had been
pregnant for about three weeks.

3. Alcoholic males were mated to alcoholic females. This is the most
severe test, both parents being alcoholic. Fourteen such matings gave in
ten cases no offspring, or very early abortions. Three still-born litters
were produced, consisting in all of six individuals, while only one living
young was born. This single offspring from the fourteen matings died
in convulsions on the sixth day after birth.

The young that have died in the experiment showed nervous disor-
ders, many having epileptic-like seizures, and all died in convulsions.

Nine control matings in the same group of animals have given nine
living litters, consisting in all of seventeen individuals, all of which
have survived and are large, vigorous animals for their ages. Two young
from non-alcoholic parents died, but this mother also died two days
later. Her diseased condition doubtless affected the suckling young.

Forty-two matings of alcoholic guinea-pigs have given only eighteen
young born alive, and of these only seven, five of which are runts, sur-
vived for more than a few weeks, while nine control matings have given
seventeen young, all of which have survived and are normal, vigorous
individuals. These facts convincingly demonstrate the detrimental
effects of alcohol on the parental germ cells and the developing offspring.

: REFERENCES

Adami, J. G.: The Principles of Pathology, Vol. I, New York, Lea and Febiger,
1908.

Ballantyne: Antenatal Pathology, Edinburgk, Green and Son, 1902.

Bertholet, E.: Ueber Atrophie des Hoden bei chronischem Alkoholismus.
Centralbl. f. allg. Path., 1909, xx, 1062.

Elderton, E., and Pearson, K.: A First Study of the Influence of Parental
Alcoholism on the Physique and Ability of the Offspring. Eugenics Lab. Memoir,
1910, x, Dulan, London.

Féré, C.: Influence du repos, sur les effets de l’exposition préalable aux vapeurs
d’alcool avant l’incubation de l’oeuf de poule. Compt. rend Soe. de biol., 1899, li;
Note sur la resistance de ’embryon de poulet aux traumatismes de loeuf. Jour.
anat. et de physiol., 1897, p. 264. Remarques sur l’incubations des oeufs de poule
privés de leur coquille. Compt. rend. Soc. de biol., 1900, lii.

Forel, A.: Alkohol und Keimzellen (blastophthorische Entartung). Miinchen.
med. Wehnschr., Dee. 5, 1911, lviii, 2596.

Herbst, C.: Experimentelle Untersuchungen, u.s.w., Ztschr. f. wissensch. Zool.,
1892, iv; Mitt. a. d. Zool. Staz. zu Naepel, 1893; Arch. f. Entwicklungsmechn. d.
Grgan., 1896, iv.

Hertwig, O.: Urmund und Spina bifida. Eine vergleichende morphologische,
teratologische Studie an missgebildeten Froscheiern. Arch. f. mik. Anat., 1892,
XXxix, 353-503.

Hodge, C. F.: The Influence of Alcohol on Growth and Development. In
Physiological Aspects of the Liquor Problem, by Billings, ed. 1, p. 359, Houghton,
Mifflin Co., New York, 1903.

a0)

Tloppe, H.: Die Tatsachen iiber den Alkohol. Ed. 3, Berlin, 1904.

Hunt, Reid: Studies in Experimental Alcoholism. U. S. Pub. Health Bull.
No. 33, 1907.

Kyrle: Bericht tiber Verhandlungen der XIII Tagung der Deutschen pathologi-
schen Gesellschaft in Leipzig. Centralbl. f. Path. u. path. Anat., xx, No. 77, 1909.

Laitinen, T.: Ueber den Einfluss des Alkohols auf die Widerstandsfiihigkeit
des menschlichen und tierischen Organismus mit besonderer Beriicksichtigung der
Vererbung. Tr. Kong. Inter. X Alkoholismus, Budapest, 1905; Ueber die Ein-
wirkung der Kleinsten Alkoholmengen auf die Widerstandsfiihigkeit des tierischen
Organismus mit besonderer Berticksichtigung der Nachkommenschaft. Ztschr. f.
Hygiene, 1908, lvili, 139.

Loeb, J.: Investigations in Physiological Morphology. III. Experiments on
Cleavage. Jour. Morph., 1892, vii, 253; Ueber die Entwicklung von Fischembryo-
nen ohne Kreislauf. Pfliiger’s Arch. f. d. ges. Physiol., 1893, liv, 525; Ueber die
relative Empfindlichkeit von Fischembryonen gegen Sauerstoffmangel und Wasser-
entziehung in verschiedenen Entwicklungsstadien. Pfliiger’s Arch. f. d. ges.
Physiol., 1894, lv, 580; Studies in General Physiology. Two volumes, Univ. of
Chicago Press, 1905.

Lustig, A.: Ist die fiir Gifte erwordene Immunitiit tibertragbar von Eltern auf
die Nachkommenschaft? Centralbl. f. Pathol., 1904, xv, 210.

Mairet and Combemale: Influence dégéneration de Valecool sur la descendance.
Compt. rend. Acad. d. Se., 1888, evi, 667.

Mall, F. P.: A Study of the Causes underlying the Origin of Human Monsters,
Jour. Morph., 1908, xix, 1-361.

MeClendon, J: F.: An Attempt Towards the Physical Chemistry of the Pro-
duction of One-Eyed Monstrosities. Am. Jour. Physiol., 1912, xxix, 289.

Morgan, T. H.: The Relation Between Normal and Abnormai Development of
the Embryo of the Frog, as determined by the Effects of Lithium Chlorid in
Solution. Arch. f. Entwicklingsmech., 1903, xvi; The Relation Between Normal
and Abnormal Development of the Embryo of the Frog. Ibid., 1902-1905, xv-xix.

Nice, L. B.: Comparative Studies on the Effects of Aleohol, Nicotin, Tobacco
Smoke and Caffeine on White Mice. I. Effects on Reproduction and “Growth.
Jour. Exper. Zool., 1912, vii, 133.

Nicloux: Passage de l’Alcool ingéré de Ja mére au foetus, ete. L’Obstetrique,
1900, xeix.

Paul, Constantin: tude sur intoxication lente par les préparations de plomb,
de son influence sur le produit de la conception. Arch. gén. de méd., 1860, xv, 513.

Pearson, K., and Elderton, E.: A Second Study of the Influence of Parental
Alcoholism cn the Physique and Ability of the Offspring. A Reply to Medical
Crities of the First Memoir. Eugenics Lab. Memoir. 13, Dulan, London, 1910.

Preyer, W.: Physiologie spéciale de ’embryon. Trad. frane., 1887, p. 16.

Simmonds, H.: Ueber die Ursache der Azoospermie. Vortr. im Aerztl. Verein
zu Hamburg, June, 1898; Berl. klin. Wehnschr., 1898, No. 36, p. 806.

Stockard, C. R.: The Development of Fundulus heteroclitus in Solutions of
Lithium Chlorid, ete. Jour. Exper. Zool., 1906, iii, 99; The Influence of External
Factors, Chemical and Physical, on the Development of Fundulus heteroclitus.
Jour. Exp. Zool., 1907, iv, 165; The Artificial Production of a Single Median
Cyclopean Eye in the Fish Embryo by Means of Solutions of Magnesium Chlorid.
Arch. f, Entwicklungsmech., 1907, xxiii, 249; 1909, vi, 285; The Origin of Cer-
tain Types of Monsters. Am. Jour. Obst., 1909; lix, No. 4; The Independent
Origin and Development of the Crystalline Lens. Am. Jour. Anat., 1910, x, 393;
The Influence of Alcohol and Other Anesthetics on Embryonic Development. Am.
Jour. Anat., 1910, x, 369.

Sullivan, W. C.: A Note on the Influence of Maternal Inebriety on the Off-
spring. Jour. Ment. Se., 1899, xlv, 489.

Todde, C.: L’azione dell’ alcool sullo svillupo e sulla funzione dei testicoli.
Riv. sper. di Freniatria, 1910, xxxvi, No. 3, p. 491.

Ziegler, H. E.: Ueber die Einwirkung des Alkohols auf die Entwicklung der
Seeigel. Biol. Zentralbl., June, 1903, xxiii, 448.

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IS THE CONTROL OF EMBRYONIC DEVELOPMENT
m PRACTICAL PROBLEM?

By CHARLES R. STOCKARD.
(Read April 19, 1912.)

Under favorable natural conditions two normal parents should,
and usually do, produce a vigorous normal offspring. When, how-
ever, the conditions of development are modified or if in the second
place the parents are not entirely normal the offspring is usually
more or less defective. I shall attempt to show that the proper
development of the offspring is dependent upon two main factors,
first the physical qualities of the parental germ cells, and second the
environment in which the embryo develops.

One is at first sight apt to think that deformities and defects
are rare among men and other animals; but closer observation will
show that the really structurally perfect individual is rather excep-
tional. Gross anatomical defects or monstrosities are frequently
found among all animals, while lesser defects of minor importance
are to be observed in a majority of individuals. These defects often
cause no inconvenience, and indeed, we may be ignorant of their
presence, since they are generally internal. Yet many apparently
normal individuals sooner or later suffer or may actually die from
some hidden developmental imperfection. The well-known con-
genital defects of the heart and other parts of the vascular system,
digestive tract, etc., as well as the numerous developmental arrests
in various parts of the body constantly remind the observer of the
great loss in ability and energy that the race suffers as a result of
faulty development.

These defects in construction must be considered a disease which
causes the death of about 23 per cent. of the human race before or
shortly after the time of birth (Sullivan’s studies and French sta-
tistics), and handicaps a certain proportion of the survivors through-

Reprinted from Proceedings American Philosophical Soctety, Vol. li, roz2.

2 STOCKARD—CONTROL OF DEVELOPMENT. | [April to,

out their lives. We carefully study and use all known precautions
to protect ourselves against post-natal infections and diseases, and
much interest and time is given to combating the causes, yet little
is said and scarcely anything done towards a control of development,
or the hygienic protection of the developing individual.

This is really a morphological problem and is as truly a part of
the fight against disease as is the treatment of abnormal physiological

processes. It is not all of morphology to describe and study the

detail of bodily structure, but its important task is to understand
and analyze that structure, and if possible control and regulate its
formation: and thus, if properly developed its goal is to relieve the
race of its great structural disease—a disease which affects more
individuals than any other one malady of man.

To most persons the above task seems at first thought a futile
undertaking, and any one suggesting such control or preventive
treatment might be interpreted as indulging in fanciful speculation.
Yet the data available from the studies of defective persons in
different countries of the world, and the experimental evidence fur-
nished by work on lower animals makes the correction or preven-
tion of developmental defects seem even today a problem to be
practically handled to a slight degree at least.

To proceed as with any other disease, we must first ascertain
the cause of these conditions, as the possibility of a cure depends
upon the nature of the cause.

Are monstrosities and defective development due to some innate
change within the germ cells of the parent, thus being incurable, as
many former workers would have us believe? Or, are they due to
changes produced in the germ cells by the action’of some unusual
condition in the body of either the male or female parent, or finally
may they not be due to an unusual environment acting upon the de-
veloping embryo itself? In both of the latter cases the conditions
are open to regulation or control. These questions may only be solved
experimentally and the experiments have proven that the great ma-
jority of monsters are due to the action of unusual conditions upon
either the parental germ cells or the developing embryo. There may be
some changes of form or variations in animals which are due to

1912. ] STOCKARD—CONTROL OF DEVELOPMENT. 3

innate changes in the germ-plasm but even these when fully under-
stood may possibly be shown to result indirectly from some change
in the chemical surroundings.

First to consider the modifications induced in the developing egg
or embryo by a strange chemical environment. It has been found
for the eggs of a number of animals that develop normally in sea-
water that when certain chemicals are added to their environment
they develop into various unusual forms.

I experimented for several years on fish’s eggs and found that
on adding any one of a large series of salts to the sea-water that
the eggs developed abnormally and gave rise to a great number of
monstrous individuals. The types of the monstrosities were vari-
able, and the same kind of monster often resulted from different
treatments. This was to be expected, but the important problem
was to produce some definite type of monster in great numbers
with any given treatment. This I finally succeeded in doing and in
some experiments got as many as 90 per cent. typical cyclopean
or monophthalmic monsters. These types of monsters first occurred
in solutions of MgCl, in sea-water. In such solutions as many as
50 in 100 eggs formed one-eyed cyclopean embryos. Since Mg
has the power to inhibit activity in animals and so acts as an anezs-
thetic I determined to try the action of a number of such substances
on the developing eggs to ascertain whether they might also inhibit
the lateral migration of eye parts. Alcohol, ether, chloroform,
chloreton, etc., were employed and cyclopean monsters resulted from
eggs developing in all of these substances. Alcohol gave the most
decided effects and inhibited the normal production of eyes in almost
all cases. All of these anesthetics act more particularly upon the
central nervous system of the adult and it is important to find that
the development of the nervous system is also especially affected
by them. In alcohol solutions the embryos showed almost every
gross abnormality of the brain which is known to occur, and the
spinal cord was often defective.

I have repeated the experiments of Féré with hen’s eggs and find
that when these eggs are exposed to fumes of alcohol many abnormal
chicks result. When hen’s eggs are placed in closed dishes over

4 STOCKARD—CONTROL OF DEVELOPMENT. [April 19,

_ evaporating 95 per cent. alcohol enough of the fumes penetrate the
shell and enter the contents of the egg to cause the developing
chick to form abnormally.

McClendon has lately found that an excess of CO, and plier
substances also cause cyclopia and brain abnormalities. Many
other workers have shown the effects of the environment on the
developing eg

esis. aE. proven that the experimenter has the power to
take an egg which would normally give rise to a perfect animal and
by proper treatment he may cause it to form a typically abnormal
individual. The monster may in many cases be able to survive and
move about. No one can question that in these experiments the
unfavorable environment modifies the form of the resulting indi-
vidual.

Does this also occur in embryos developing in the mother’s body?
Children are born which exhibit the same types of deformities as
those described above. Syphilitic mothers usually abort or give
birth to abnormal children and there is much evidence to indicate
that an alcoholic mother is more apt to produce an abnormal child
than is a non-alcoholic mother.

Tubal pregnancies are common among women with venereal dis-
eases and in such cases the embryo must necessarily develop under
abnormal environment, having a poor surface for placental attach-
ment in a region not adapted to the conditions of pregnancy. The
conditions for embryonic nutrition are poor. Mall has found that -
while only 7 per cent. of uterine pregnancies in his records con-
tained pathological embryos, that 96 per cent. of the embryos in
tubal pregnancies were pathological, only 2 in 46 specimens being
normal. This is strongly indicative of an abnormal environment as
the cause of abnormalities. If these monsters were due to inherent
tendencies in the germ cells one should not expect more abnormal
tubal than uterine embryos.

Among lower mammals it has been shown that dogs fed on
alcohol produce deformed and otherwise defective pups. I am now
conducting a series of experiments with guinea pigs which show
that a female treated with alcohol during her pregnancy will often

1912. ] STOCKARD—CONTROL OF DEVELOPMENT. 5

abort or produce defective young, while the control animals are
giving birth to normal young. Many more cases could be cited if
time permitted.

Experiments on lower animals, therefore, show and human sta-
tistics seem to indicate that the cause of structural disease is often
an abnormal developmental environment. To prevent such a disease
the developmental conditions must be controlled and rendered as
nearly normal as possible.

The second consideration is whether abnormal chemical environ-
ment may act on the parental germ cells in such a manner as to
cause them to change and become incapable of giving rise to a normal
individual. It is well known that certain disease toxins such as that
of syphilis and substances such as alcohol and lead effect various
body tissues so as to render them unfit for normal physiological
activity. It is, therefore, only logical to suppose that the same or
similar substances may effect the germ cells and so derange their
chemical constitutions as to cause them to give rise to offspring of
peculiar structure and qualities.

Bertholet has found that alcoliol has a particular affinity for the
reproductive glands just as it does for the nervous system. In
examining the structure of the testicles from a large number of
chronic alcoholics it was shown that spermatozoa were absent en-
tirely or degenerate in form (azoospermy) in a majority of the
cases. It is doubtless true that the ability of the spermatozoa to
accomplish normal fertilization would be affected long before any
definite structural change could be observed.

The crucial case is the treatment of the male in such a way as
to render his spermatozoa unable to produce a normal development
when combined with a healthy egg from a normal female. In this
case the action must of necessity be on the germ cell only and not
on both the egg and embryo as it might be in treating a female
mammal.

It must be recognized that an individual owes its structure and
character to the peculiar chemical constitution of the germ cells
from which it arises. The germ cells of two species of animals are

6 STOCKARD—CONTROL OF DEVELOPMENT. [April 19,

probably as different chemically as the animals are morphologically.
Therefore, if the chemical nature of the germ cells is disturbed or
injured by the action of poisons in the animal's blood they will prob-
ably show this injury in the type of individual to which they
give rise.

Constantine Paul long ago found in studying 88 cases of preg-
nancy among women lead workers that 71 resulted in abortion, pre-
mature labour, or stillbirth while only 17 children were born alive
and of these five died within the first year. Several of these women
later produced healthy children after leaving this work. (This indi-
cates that -when the cause is known for defective development the
cure may often be established by its removal.) Lead not only
effects the developing foetus but also acts directly upon the germ
cells as is shown in the case of men working in lead while their
Wives were not exposed to the poison. Many of the offspring from
such fathers are aborted and the children born are epileptic, feeble-
minded or generally defective.

To return to the results furnished by the guninea pig experi-
ments referred to above—I have chosen healthy individuals and
treated them daily with the fumes of 95 per cent. alcohol to about
the point of intoxication. Feeding alcohol and giving it by stomach
tube was first tried, both of these methods were unsatisfactory as
the guinea pigs did not take alcoholic food in sufficient quantity and
the stomach tube disturbed the animals to such a degree that I
feared the experimental result might be vitiated even though it
could be partially controlled. The inhalation method is perfectly
satisfactory ; the animals are placed in a copper tank having a screen
floor which holds them above the evaporating alcohol. The alcohol
is breathed directly into the lungs and affects the animals readily,
in much the same manner as weak treatments of ether or chloroform
would. The animals are thus put into a condition of chronic alco-
holism, being almost intoxicated six times per week. Many of these
guinea pigs have been killed and their lungs, liver and other organs
examined and found to be perfectly normal so far as their appear-
ance goes. The conjunctiva over the eyes is very often affected
by the fumes, during the beginning of the treatment the eyes often

1912. ] STOCKARD—CONTROL OF DEVELOPMENT. r

become white, this is transitory in most instances and the eyes finally
clear again and remain in a normal condition from then on. Most
of the specimens have fattened under the alcohol treatment.

The matings have been made in such a fashion as to test several
questions. First, alcoholic males are mated with normal females,
paternal influence, the crucial test for the effect upon the germ cells.
Second, alcoholic females are paired with normal males, the maternal
influence plus the direct action on the developing embryo. Lastly,
alcoholic males and females are paired.

The results of 40 such matings are shown in Table I. The
decided effects of the alcoholic treatment are seen when the records
are compared with those of the normal guinea pigs.

TABLE. I.

MatTINGs oF ALCOHOLIZED GUINEA-PIGS.

Num | pec! san | Num | oS eee
Condition of Animal. | Le as lor Early, born ber of | Living Early Deaths. SEENEDS
elatos enon rl baters Still- | Litters. | Young.
| ings. Gone | * | Born.
|
Alc. male X
nor. female ..... 24 | 14 5 8 5 5 5
Hapa!
Nor. male X alc.
femmalernys sie): Dea tr il) oe ) ie r) I
| Preg. 2 in
» utero, I de-
formed.
Alc. male X alc.
Hee yy Algo I4 m0) ae. 5 i || I fe)
died 6th day
STMAMIATY; 6 oso 40 25 8 13 7 6 - 6
6 in 25
Nor. male X nor.
female. Control. 8 Onl on || oO 8 oO 15
| | 15 in 15

In the 24 cases in which normal females were mated with alco-
holic males, 14 gave negative results. Some of these probably
aborted early as the parents were all fertile and the female is apt to
eat the young before they have been observed when they are born
prematurely. Five of the matings gave stillborn young, in some
cases they were born a little before term. Litters were born alive but
the young died soon after showing many nervous symptoms, such

8 STOCKARD—CONTROL OF DEVELOPMENT. [April 19,

as epileptic-like seizures, and all died in convulsions. Only two
litters consisted of normal offspring and these young, five in all, seem
healthy though unusually small. It is thus seen that in 24 matings
of normal females with alcoholic males only two gave normal results.
Whereas in the control animals all matings have resulted in the pro-
duction of normal offspring.

Only two matings were made between normal males and alco-
holic females. One of these gave no result or was possibly aborted
very early and lost, while the other mating produced one female off-
spring that lived to become pregnant by an alcoholic male. This
last mentioned female was killed by accident, two embryos were
found im utero one of which was deformed.

Fourteen matings were made between alcoholic males and
females. Ten gave no result or aborted very early and were eaten,
while four cases showed the following records. One young was
born weak and died in convulsions on the sixth day after birth.
Two cases of premature births of dead young. One female had

young im utero when killed.

TABLE IL

SuccessivE MATINGS OF TEN FEMALES.

Animal, ist Mating. | 2d Mating. 3d Mating. 4th Mating.

No. 10 ale. |Ale. male 4=1, |Alc. male 4=0. |Alc. male 6=o0. |Alc. male 4=1,

young died 6th | embryo in u-
day. tero 2nd week.

No. 12 alc. Alc. male 5=o. |Alc. male 5 =o. |Ale. male 4=0. |

No. 11 alc. Alc. male 6=o0. |Alc. male 6=0. |Alc. male 5 =2, |Alc. male 4=o0.
premature,

still-born.

No. 13 nor. |Alc. male 5 =1, |Alc. male 5 =o. |Alc. male 4=0.

stillborn. |

No. 17 nor. |[Eth. male 1 =o. |Eth. male 1 =o.

No. 18 nor. ‘Alc. male 5 =o. Alc. male 5 =o. |Alc. male 6 =o.

No. 7 nor. Eth. male 2=2, /Eth. male 2=o0. |Eth. male 2 =o.
| premature, |
| stillborn. | !

No. 14 nor. ‘Eth. male 3 =o. Eth. male 3 =o.

No. 19 nor. '!Alc. male 4=0. |Alc. male 6=1, |Alc. male 6=o. |Alc. male 5 =4,
| | stillborn. small but ac-
| tive.

No. 15 nor. \Ale. male 6 =2, Alc. male 5=o0. |Alc. male 5=2, |Nor. male =1,
| stillborn. died 4th week.| normal young.

1912.] STOCKARD—CONTROL OF DEVELOPMENT. 9

These results stand in marked contrast to the records of the
control, which show all normal conceptions and normal offspring.

The second table shows the results of successive matings in ten
of the females. The varying success of the conceptions in the same
individual are striking.

Nice has quite recently recorded a similar series of experiments
with alcohol on mice. Alcohol was given to the mice in their food.
Nice finds that while there was a certain fatality among the offspring
from alcoholic parents as compared with those from normal parents,
where there was no fatality, yet nevertheless the offspring of alco-
holic parents actually grew faster than those from the control. This
may indicate that alcohol is not equally poisonous in its effects upon
all animals, as might really be expected. The germ cells of mice
may be more or less immune to the action of alcohol. It is well
known that the action of alcohol is different in its effects on indi-
viduals from different human families. Some alcoholics show
chiefly nervous disorders, hallucinations, delirium, etc., while others
may have no nervous symptoms but exhibit various derangements
of the digestive glands, kidneys, etc., or may have a fatty degenera-
tion of almost all organs.

Finally it may be concluded that the experimental evidence goes
to show that the development of an offspring may be modified by
either treating the parents so as to affect their germ cells or by
subjecting the developing embryo itself to unusual or injurious
conditions.

The causes of many congenital defects are therefore known. It
is possible to control embryonic development to such an extent as
to produce abnormal structures. May not the proposition be re-
versed and unfavorable environments be treated in such a manner
as to render them favorable to normal development? Diseased
mothers may in some cases, at least, be made fit for the function
of reproduction.

The regulation of structural disease becomes then a problem of
morphology and hygiene. It is most important, and must precede,

10 STOCKARD—CONTROL OF DEVELOPMENT. _ [April 19,

or go before, the selective mating of human beings or the eugenics
movement. The most intellectual will rarely submit to direction in
choosing a mate, yet every productive pair will welcome any possible
means of improving the quality of their offspring.

While preventive measures are being used to protect the post-
natal life of the individual, why not guard as far as possible its pre-
natal development ?

CorNELL University MeEpIcaL COLLEGE,

DEPARTMENT OF ANATOMY,
New York CIty.

1!

m

fi

'

An Experimental Study of the Influence of Alcohol on
the Germ Cells and the Developing Embryos of Mammals.

By
Charles R. Stockard and Dorothy M. Craig.

(Anatomical Laboratory, Cornell University Medical College,
New York City, U.S. A.)

Eingegangen am 25. Juni 1912.

The eggs of lower vertebrates which develop outside the ma-
ternal body may be induced to give rise to abnormal or monstrous
forms when subjected to various unusual conditions. By regulating
the chemical environment in which the eggs develop definitely typical
defects may be produced in great numbers. The same type of defect
may be caused by a variety of substances. This fact was shown
by Srockarp ‘10 in studies on cyclopia and also recently by
McCLENDON in similar experiments. The occurrence of the same
defect under various conditions might be expected and has often been
observed by numerous workers.

The important fact, however, is that under certain conditions a
particular defect can be made to occur in a large number of the
embryos.

In some of the alcohol experiments (Stockarp '10) on fish’s
eggs dozens of embryos with typical brain and eye defects occurred
while few or no other types of deformities existed. The experi-
menter has the power in these cases to predict, with at least a
limited degree of certainty, the type of deformity which will result
from a definite intensity of a certain treatment. Embryonic develop-
ment in such cases may be partially regulated or controlled.

BarDEEN ‘07 showed that when normal eggs of the toad are
fertilized by spermatozoa which have been modified by treatment

Archiv f. Entwicklungsmechanik. XXXY. 37

570 Charles R. Stockard and Dorothy M. Craig

with the Roentgen Rays the eggs develop abnormally. O. Hertwie ‘11
has since recorded similar results by treating the spermatozoa of frogs
with radium. These experiments show convincingly the influence of
the male germ cell on the development and structure of the embryo.
Thus in the lower vertebrates it has been possible to modify develop-
ment by treating either the egg or spermatozoon.

The question now arises whether the germ cells and develop-
ing embryos of mammals may be similarly affected. The conditions
in mammals are more complex since the embryo develops internally,
and the substances administered to the body of the parent may in-
duce secondary effects which will modify or confuse the results.
Should a substance be secured that will act directly upon the germ
cells or the tissues of the developing embryo within the parental
body, then one might expect to regulate the action of this substance
in much the same manner as in experimenting upon eggs develop-
ing in sea-water. From a number of recent observations it would
seem that alcohol is just such a substance as is required.

It is an accepted fact that alcohol may cause changes and de-
generation in many of the body tissues. How then can the repro-
ductive tissues escape, or may they be affected without seriously
injuring other tissues? Brrruotet, 1909, made an extensive ex-
amination of the influences of alcohol upon the histological structure
of the reproductive glands and found much degeneration and atrophy
in the testicular parenchyma of chronic alcoholics, both young and
old. NicLtoux has shown that alcohol has a marked affinity for the
reproductive glands and that alcohol may occur, as such, in the
testicular tissues and in the seminal fluid of mammals. The ripe
spermatozoa may, therefore, be bathed in a weak solution of alcohol
shortly before fertilizing the egg, and if affected by the alcohol the
spermatozoon may cause the developing eggs to give rise to a de-
fective or deformed individual.

Nictoux has also demonstrated the passage of alcohol from
the blood of the mammalian mother into the tissues of the embryo
(guinea-pigs and dogs). After a short period of time the amount of
alcohol in the blood of the foetus is about equal to that in the blood
of the mother, while there is really more alcohol in a given weight
of foetal tissue than is to be found in an equal weight of liver
tissue from the mother. The reality of the passage of alcohol from
the mother to the foetus demonstrates the possibility of the intoxi-
cation of the foetus.

An Experimental Study of the Influence of Alcohol ete. 571

The experiments on mammals do not then really differ greatly
from those on the lower vertebrates, where the externally déyelop-
ing eggs may be placed directly in various unusual solutions; since
the egg or embryo, although within the mother’s body, is readily
bathed or impregnated by the alcohol contained within the mother’s
blood.

The few experiments upon the influence of alcohol on the mam-
malian offspring are not at all conclusive and are somewhat contra-
dictory.

CoMBEMALE and Hopce studied the effects of aleohol upon the
offspring of dogs and recorded injurious effects, though their experi-
ments were performed on very limited numbers of individuals.

Quite recently Nice has recorded experiments which seem to
show that alcohol exerts a very slight effect upon the offspring of
mice. He has given small doses of alcohol mixed with the food
and also supplied 35°) alcohol instead of water for the animals to
drink. Much of the alcohol probably evaporated from the food be-
fore it was eaten. Animals treated in this way gained in weight
over the control, and their offspring excelled the control in rate of
growth. The fecundity of the treated mice was greater than that of
the control but there was also a greater mortality of the offspring
from the treated parents. None of the control young died while
11.1% of the alcoholic young died soon after birth. There were no
abortions, no still-births and none of the young were deformed.

Mice may possibly be peculiarly resistant to alcohol though we
should rather think that they received too little to give a pronounced
effect, yet it was sufficient to cause a certain fatality among the
offspring.

The few studies on mammals have failed to produce convincing
evidence of the specific actions of alcohol. Yet the statistical data
from observations on defective human beings would indicate that
alcohol had a special affinity for the developing nervous system‘).
The experiments upon the influence of alcohol on the developing
fish embryo (Srockarp ‘10) demonstrated that aleohol did have a
specific affinity for the central nervous system and caused the brains
of these embryos to exhibit numerous deformities while the organs
of special sense were also affected.

1) The senior author has given a somewhat extensive review of the literature
pertaining to this subject in the Archives of Internal Medicine, 1912.
37*

572 Charles R. Stockard and Dorothy M. Craig

Method and Material.

The experiments here recorded were undertaken in order to
ascertain whether alcohol did exert a marked influence on the germ
cells and developing embryos of mammals, and if possible to de-
monstrate the nature and mode of action of this influence. Alcohol
was employed as an agent since it may be given to guinea-pigs
without greatly disturbing their normal physiological processes and
so does not produce marked conditions which would secondarily effect
the results. As before mentioned alcohol may remain as such in the
blood and tissues of a mammal and may thus act directly upon the
embryo just as it would when added to the sea-water in which fish’s
eges were developing. It is an active substance, and, therefore,
lends itself admirably to experimental use.

The experiments have been conducted on guinea-pigs since they
breed fairly rapidly and rear their young without much difficulty in
the laboratory. Strong healthy stock has been chosen and the ani-
mals have been carefully handled. All have remained in vigorous
health and most of them have increased in size and fattened dur-
ing the progress of the experiment. The males and females have
been kept separated and individual pairs were mated from time
to time.

The animals are first tested by normal matings and found to
produce normal offspring. The alcoholic treatment is then begun on
a given number of individuals and the males and females mated in
different combinations according to whether they are alcoholics or
normal. An alcoholic male is mated with a normal female, the
paternal test, this is the crucial test of the influence upon the germ
cells as here the defective offspring must be due to the chemically
modified spermatozoon from which it arises, since the egg and the
mother in which the embryo develops are both normal.

Normal untreated males are paired with alcoholic females, ma-
ternal test, in this case the defective offspring may be due either
to a modified ovum or to the fact that it developed in a mother with
alcoholic blood, therefore supplying an unfavorable developmental
environment. Lastly, its condition may be due to both of these
causes. The mammalian mother has two chances to injure an off-
spring, either by producing a defective egg or secondly by supply-
ing an unfavorable or diseased environment in which the embryo
develops.

An Experimental Study of the Influence of Alcohol: ete. 573

The final combination is the mating of alcoholic individuals, this
of course offers the greatest chance for defective offspring.

Alcohol is administered to the animals by inhalation. It was
first given with the food, but the animals did not relish it, and there-
fore took less food. It was then given by stomach tube but this
method so upset the animals that the results might have been modi-
fied by their poor bodily conditions and the bad state of their sto-
machs. The inhalation method is entirely satisfactory, the guinea-
pigs thrive and usually gain in weight during the experiment, they
have good appetites and are in all respects apparently normal. The
only indication of the effects of the treatment is shown by the quality
of offspring they produce.

The apparatus used for giving the alcohol consists of an air
tight copper tank 36 inches long by 18 inches wide and 12 inches
deep with a sloping bottom draining to the center. Over this bottom
is placed a wire screen and below the screen cotton soaked with
95°/, alcohol is spread. The tank is closed and allowed to stand
until the atmosphere within is saturated with alcoholic fumes. A
ventilation system is so arranged that a given quantity of alcohol
fumes may be driven through the tank in a given time, but it has
not seemed advisable to use this device as the degree of intoxication
is a better index to the physiological response of the animals. Their
resistance to the fumes is changeable. The guinea-pigs, three or
four at the time, are placed on the wire screen above the evaporat-
ing alcohol, the tank is again closed and the animals are allowed
to remain until they begin to show signs of intoxication, though they
are never completely intoxicated. They usually inhale the fumes for
about one hour. The animals are treated in this way for six days per
week and some have now been treated over a period of about nineteen
months. None of the effects are due to want of air since the same
number of guinea-pigs may remain for hours in this closed tank without
showing any signs of discomfort when there are no fumes present.

In order to avoid handling the females during late pregnancy
a special treating cage is devised for them. An ordinary box run
with a covered nest in which the animal lives is connected by a
drop door with a metal lined tank having a similar screen arrange-
ment to that described for the general treatment tank. The pregnant
animal may be driven daily into the tank and thus treated with alcohol
fumes throughout her pregnancy without having to be handled or moved
about in any way that would tend to disturb the developing foetus.

Charles R. Stockard and Dorothy M. Craig

on
=~]
TS

Results.

During the vapor treatment the animals usually react in a manner
quite similar to their behavior in weak fumes of ether or chloroform.
The majority of them sit quite motionless and sniff their noses for
atime and then become somewhat drowsy. A few individuals, how-
ever, are excited by the treatment and run about the tank becoming
sexually excited and may often fight other animals savagely. One
of the males fights and bites so vigorously while taking the fumes
that he has to be treated separately from all others. The fumes thus
have a different influence upon the behavior of different individuals
in much the Same way that alcoholic intoxication expresses itself
differently on different human beings.

During the first few weeks of the treatment the fumes cause
the eyes to water so that tears run over the face. The nose and
mouth also become moist and the animals sniff almost constantly.
Alcoholic fumes are very irritating to the mucous membranes at first.
The conjunctiva of the eye becomes irritated and finally opaque in
some instances, so that the eye takes on a white appearance. The
tissues seem, however, to develop a resistance to the fumes. The
eyes become clear after a few months and never again become opaque.
The nasal mucosa also ceases to secrete excessively unless the ani-
mal is left in the tank for an unusually long time.

Many of the guinea-pigs have been killed after treatments of
different duration up to fifteen months and all of their viscera care-
fully examined and the reproductive glands studied microscopically.
In no case have we found any changed structures due to the alco-
holic treatment. The lungs, liver, stomach, intestines, kidneys, repro-
ductive glands, brain and all other parts appear perfectly normal.
The general health and behavior of the animals also indicate that
they are in good condition. As before mentioned several animals
have been partially castrated during the experiment. One of the
reproductive glands was removed and examined microscopically. In
all cases the germ cells, ova or spermatozoa, were found to exhibit
perfectly normal structure. One cannot claim, therefore, that this
treatment is excessively severe or greater in proportional amount
than the alcohol a human being often takes. The matter of fact is
that these animals have never been completely intoxicated but receive
only enough alcohol six times per week to affect their nervous states.

An Experimental Study of the Influence of Alcohol ete. 575

They may be compared to a toper who drinks daily but never be-
comes really drunk.

While the bodies of these animals show no direct effects of the
alcohol, the conditions of the offspring to which they give rise
exhibit most strikingly the effects of the alcoholic treatment. The

results of mating the alcoholized guinea-pigs are summarized in
Table I.

Table I.
Effects of Alcohol on the Offspring of Guinea-Pigs.

_ Condition peer SUN ae hake eae oe ee al taieete) || OEE OO ervey
2 Sree a) O8 early | litt still-born | litt dying soon | i
| 6 abortion | ae young re after birth | O"7S
Alcoholic male by |
normal female . 24 14 5 8 5 7 51)
Normal male by |
alcoholic female 4 1 ) 0 = 3 2 2)
iL.
Alcoholic male by | aah
alcoholic female | 14 10 3 6 Ligh 1 0
aor | 6th day
Summary ....| 42 | 25 | 8 14 9 13) byez
Normal male by | |
normal female . 94) 0 0 0 9 | 0 Ce
Control |

Fifty-five matings of treated animals have been made. Forty-
two of these have now reached full term and are recorded. Thirteen
matings are not yet due. From the forty-two matings only seven
young survived, and six of these are still living, five of which are
small for their ages though their parents were unusually large strong
animals.

1) Four survivors in one litter, and one was a member of a litter of three,
the other two died immediately after birth.

2) One lived to become pregnant with two young in utero, one deformed.
Other survivor normal, the mother was not treated until after first two or three
weeks of pregnancy.

8) Of thirty-two young born only seven have survived.

4) One other non-alcoholic mating was made from which two young re-
sulted, they died after the second and fourth days respectively and the mother
died two days later, her diseased condition no doubt affected the suckling young.
They have for this reason not been included in the normal control.

576 Charles R. Stockard and Dorothy M. Craig

The conditions of the animals in the mating pairs are shown
in the first column of the table and the total results of the mating
are indicated in the following columns.

The first horizontal line gives the records when alcoholic males
are paired with normal females. Twenty-four such matings were
made. Fourteen of these gave negative results or resulted in early
abortions. Many embryos were aborted during very young stages
and some of these were deformed, though they were generally in
such poor conditions after being cast out into the cages that little
could be learned from them. They were partially or completely
eaten by the mother in most cases. The males were always kept
for a number of days with the females during favorable periods and
conception should have occurred in all cases, as it did in the control
matings. Only ten of the twenty-four matings resulted in concep-
tions which ran the full term. Half of these, or five, were still-born
litters. There were three still-born litters of two young each and
two of one individual each. Most of these were slightly premature,
their eyes being closed and the hair sparce on their bodies. (A
normal guinea-pig at birth is well covered with a hairy coat, its
eyes are open and it very quickly begins to run about actively.)

Five litters of living young were born. One litter consisted of
only one young, a weak individual that grew very little and died
after six weeks. Two litters contained two young each. The mem-
bers of one of these litters died during the first and fourth weeks
having been weak and small since birth. Both of those in the other
litter were in a similarly feeble condition and died before the first
month. One litter contained three young, two of these died immedi-
ately after birth; the other one is still alive though small for its
age. The fifth litter contained four young all of which are runty
though their parents were unusually large animals. Thus out of
twenty full-term young of which only twelve were born
alive but five individuals have survived and these are un- .
usually small and very shy and excitable animals.

It is a point of some interest that all of the young animals that
died showed various nervous disturbances having epileptic-like seiz-
ures and in every case died in a state of convulsion.

The important fact in the above cases is that only the father
was alcoholic, the mother being a normal vigorous animal. This
experiment clearly demonstrates that the paternal germ
cells of mammals may be modified by chemical treatment

An Experimental Study of the Influence of Alcohol ete. 577

to such a degree that the male will beget abnormal off-
spring even though he mate with a vigorous female. ‘A re-
consideration of the figures in the first line of the table shows really
how decidedly the injured spermatozoon expresses itself in the fate
of the egg with which it combines.

The second line of the table shows the results of matings be-
tween alcoholized females and normal males. These matings might
be expected to give more marked results than the previous ones,
since in the treated females not only the germ cells may be affected
but the developing embryo itself may be injured by the presence of
alcohol in the blood of the mother. NicLtoux has shown that alco-
hol may pass directly from the maternal blood into the embryonic
tissues of a guinea-pig.

The spermatozoon, however, is probably a more sensitive struc-
ture than the egg and is easily injured or killed by slightly abnormal
conditions. It might possible be that when such specialized cells
swam for even a short time in seminal fluid containing a trace of
alcohol that their chemical nature would be so decidedly disturbed
as to render them incapable of inducing normal development after
impregnating the eggs. At any rate the few cases at present avail-
able seem to indicate that the effect on the offspring is equally as
great when it is produced by an alcoholic father as by an alcoholic
mother.

There are only four matings between alcoholized females and
normal males. One of these gave a negative result or was possibly
aborted very early. Three living litters were born. One litter con-
sisted of three premature young which died shortly after birth. The
remaining two litters each contained only one young but these two
animals survived. One of these guinea-pigs was born after the mother
had been treated for three and one half months. The offspring was
weak and small for several months after birth but finally recovered
and developed into a normal animal. This animal, a female, was
mated with an alcoholic male and became pregnant. Unfortunately
she was killed by accident and on examination her uterus was found
to contain two embryos of 33 and 32 mm. in length. One of these
embryos was deformed and showed very decidedly degenerate and
feebly developed hind legs. The posterior end of its body was also
poorly formed. This is of interest since all of the affected offspring
of alcoholic guinea-pigs are weak in their hind extremities and drag
their legs. Yet none were so modified as to show a noticeable

578 Charles R. Stockard and Dorothy M. Craig

structural defect except this embryo, which had one alcoholic grand-
mother and an alcoholic father.

The only other survivor from an alcoholic mother is strong and
full grown for its age. The mother had been treated for only one
and one-half months when the offspring was born so that she was
normal during the first two or three weeks of pregnancy. No doubt
the early stages of development are more easily modified to produce
significant defects than are the later. This question is being more
fully tested on guinea-pigs with experiments now in progress.
STOCKARD has shown, however, in treating fish’s eggs that the
period at which the treatment is applied is a most important factor
in determining the type of defect or modification which will result.
Certain salts, different strengths of magnesium chloride for example,
which give pronounced effects when added to the sea-water con-
taining eggs in early developmental stages may really be ineffective
after the eggs have developed beyond these stages. In the case
under consideration the offspring might not have fared so well if
the alcoholic treatment had been started on the mother a few weeks
before conception, instead of three weeks after her pregnancy had
begun. This with other points shall be more completely analyzed
in future communications on these experiments.

The four matings of alcoholic females and normal males resulted
in three living litters consisting in all of five individuals. Three of
the young were premature and died shortly after = while two
young survived.

Finally, we may consider the results of pairing two alcoholized
individuals. The third line of the table summarizes these data. As
might have been anticipated this type of mating has given the highest
fatality of all.

Ten out of a total of fourteen matings have given no offspring
or early abortions which were in many cases eaten by the mother.
Three still-born litters have been produced each consisting of two
young. Only one living litter was born from the fourteen
matings in which both parents were alcoholic and this lit-
ter consisted of but one weak individual which died in con-
vulsions on the sixth day after birth. This is indeed a decided
effect of alcohol upon the offspring when one considers the nine
control matings, all of which gave living litters containing a total of
seventeen individuals all surviving.

Two young which were included in the sole and died, should

An Experimental Study of the Influence of Alcohol ete. 579

not really be counted. They died four days after birth and the
mother died two days later in a diseased condition. No doubt the
poor state of the mother had much to do with the fate of the suck-
ling young. She was an animal that had only been in the experi-
ment for a short time (this was her first mating) and is one of the
very few that have contracted disease or died during the nineteen
months of the work.

The fourth line of Table I gives a summary of the experiments.
There have been forty-two full-term matings, twenty-five of which
gave no offspring or early abortions, eight still-born litters have
occurred consisting of fourteen individuals, only nine living litters
have been born, 21°/, of the matings, these contained eighteen young
and but seven of this number have survived and five of these sur-
vivors are runts or small for their ages.

The bottom line of the table shows nine control matings, all
have given living litters containing a total of seventeen young, all of
them surviving. Two young that died, as stated above, were from
a dying mother and are not included in the control.

Records of successive matings of ten of the female guinea-pigs
are shown in Table Il. The varying ways in which the same indi-
vidual has responded in different matings is noticeable. Number 10,
an alcoholic female, first mated with an alcoholic male gave one
young which died on the sixth day after birth. On being remated
with the same male No. 10 gave no offspring. Then mated with
another alcoholic male gave no offspring. She mated again after
several months with the first male and on being killed was found
to contain one embryo in utero about two weeks old.

Female No. 15, a normal guinea-pig, shows an instructive record.
She was mated with an alcoholic male and gave birth to two still-
born young. Mated to another alcoholic male and gave negative
result. Remated with the second male and gave two young both of
which died of convulsions in four weeks after birth. She was then
mated with a normal male as a control and gave one vigorous nor-
mal offspring which survived.

The other records are easily understood.

These experiments have suggested many questions still to be
solved, some ot which are now being tested. The length of time
necessary to treat an animal before the resulting offspring is affected,
whether this time is equally long for both sexes, and what amount
of individual variation may exist. An important point to ascertain

580 Charles R. Stockard and Dorothy M. Craig
Lables
Successive Matings of 10 Females.
Animal ist Mating 2nd Mating 3rd Mating 4th Mating
No. 10 Ale. | Ale. male 4, | Ale. male 4 Ale. male 6 Ale. male 4,
1 young died| 0 0 1 embryo in
in 6 days utero, 2 weeks
after
No. 12 Ale. | Ale. male 5 Ale. male 5 Ale. male 4
0 0 0
No. 11 Ale. | Alc. male 6 Ale. male 6 Ale. male 5 Ale. male 4
‘ 0 0 2 premat. 0
still-born
No. 13 Nor. | Ale. male 5 Ale. male 5 Ale. male 4
1 still-born 0) 0
No. 17 Nor. | Etherized male 1) Etherized male 1
0 0
No. 18 Nor. | Ale. male 5 Ale. male 5 Ale. mae 6
0 0
No. 7 Nor. Etherized male 2) Etherized male2) Etherized male 2)
2 premat. still- 0 0
born
No. 14 Nor. | Etherized male 3) Etherized male 3
0 0
No. 19 Nor. | Ale. male 4 ' Ale. male 6 Ale. male 6 Ale. male 5
0 1 still-born 0 4 small, active
only 1/9 size,
| but living
No. 15 Nor. | Ale. male 6 Ale. male 5 ‘| Ale. male 5 Nor. male
2 still-born 0 2 died 4th wk.| 1 normal vigo-
convulsions rous young

is whether the effects of the alcoholic treatment are permanent, or
does the animal recover after a time and again become capable of
giving normal offspring. One of the most valuable problems is to
regulate the treatment in such a manner as to induce a definite type
of defect with a given kind or degree of treatment. The structure
or morphology of the monsters and defective offspring which occur
is to be carefully studied. Many other points might readily be
suggested.

a es

An Experimental Study of the Influence of Alcohol ete. 581

Definite and well controlled experiments with alcohol and other
Substances on the mammalian offspring have not been sufficiently
studied. The work is really in its beginning, and while there is
much evidence to show that various toxic agents do effect and modify
the offspring, facts are badly needed to demonstrate the regularity
and manner of this modification. The present experiments seem to
us to demonstrate in a convincing way that alcohol may readily
effect the offspring through either parent, and that this effect is al-
most fatal to the existance of the offspring when the parents have
been treated with even fairly large doses of alcohol. Many of the
cases seem to indicate further, that the tissues of the nervous system
in the offspring are particularly sensitive in their responses to the
induced conditions.

Summary.

Guinea-pigs have been treated with alcohol in order to test the
influence of such treatment on their offspring. Male and female
animals are given alcohol by an inhalation method until they begin
to show signs of intoxication, though they are never completely in-
toxicated. They are treated for about one hour at the time, six days
per week. The treatment in some of the cases has now extended
over a period of nineteen months. The animals may be said to be
in a state of chronic alcoholism.

Fifty-five matings of the aleoholized animals have been made,
forty-two of which have reached full term and are recorded.

From these forty-two matings only seven young animals have
survived, and five of them are unusually small though their parents
are large vigorous guinea-pigs.

The following combinations were made:

1) Alcoholic males were mated to normal females. This is the
paternal test, and is the really crucial proof of the influence of al-
cohol on the germ cells, since the defective offspring in this case
must be due to the modified spermatozoa, or male germ cells, from
which they arose. Twenty-four matings of this type were made,
fourteen of which gave no offspring or very early abortions; five
still-born litters were produced consisting of eight individuals in all,
and five living litters containing twelve young. Seven of these twelve
died soon after birth and only five have survived. Four of the sur-
vivors are from one litter and the fifth is the only living member
of a litter of three.

582 Charles R. Stockard and Dorothy M. Craig

i

2) Normal males were mated to alcoholic females. This is the
maternal test, in such cases the alcohol may affect the offspring in
two ways, by modifying the germ cells of the mother or by acting
upon the developing embryo in utero. Only four such matings were
tried. One gave no offspring, three living litters were born, one
consisting of three premature young that died at birth, while the
other two consisted each of one young which has survived. The
alcoholic treatment in one of the last cases was only begun after the
mother had been pregnant for about three weeks.

3) Alcoholic males were mated to alcoholic females. This is the
most severe test both parents being alcoholic. Fourteen such matings
gave in ten cases no offspring or very early abortions. Three still-
born litters were produced consisting in all of six individuals, while
only one living young was born. This single offspring from the
fourteen matings died in convulsions on the sixth day after
birth.

The young that have died in the experiment showed nervous
disorders many having epileptic-like seizures and all died in con-
vulsions.

Nine control matings of the same group of animals have
given nine living litters consisting in all of seventeen individuals,
all of which have survived and are large vigorous specimens for
their ages.

Fourty-two matings of alcoholic guinea-pigs have given
only eighteen young born alive and of these only seven,
five of which are runts, survived for more than a few weeks,
while nine control matings gave seventeen young allof which
have survived and are normal vigorous individuals. These
facts convincingly demonstrate the detrimental effects of al-
cohol upon the parental germ cells and the developing off-
spring.

Zusammenfassung,

Meerschweinchen wurden mit Alkohol behandelt, um den Einfiu8 einer
solehen Behandlung auf ihre Nachkommenschaft zu erweisen. Miinnliche und
weibliche Tiere bekommen Alkohol mittels einer Inhalationsmethode, bis sie
Zeichen von Intoxication aufweisen, doch werden sie niemals véllig betrunken
gemacht. Sie werden jedesmal etwa 1 Stunde lang an 6 Tagen der Woche
behandelt. Die bisherige Behandlung erstreckt sich in einigen der Fille auf
eine Dauer von 19 Monaten. Die Tiere befinden sich sozusagen in einem Zu-
stande von chronischem Alkoholismus.

An Experimental Study of the Influence of Alcohol ete. 583

Es wurden 55 Paarungen der alkoholisierten Tiere vorgenommen, von
denen 42 bis zum vollen Schwangerschaftsablauf kamen und hier benutzt sind.
Von diesen 42 Paarungen sind nur 7 junge Tiere am Leben geblieben, und 5
von diesen sind ungewiéhnlich klein, obgleich ihre Eltern groBe, kriftige Meer-
schweinchen sind.

Die folgenden Kombinationen wurden versucht:

1) Alkoholische Miinnchen wurden mit normalen Weibchen gepaart. Das
ist also die Priifung des viiterlichen Einflusses und stellt den entscheidenden
Versuch betreffs des Einflusses des Alkohols auf die Keimzellen dar, da in
diesem Falle die beeinfluGten Spermatozoen oder miinnlichen Keimzellen die Ur-
sache der defekten Nachkommenschaft sein miissen, von denen sie ihren Aus-
gang nimmt. 24 Paarungen dieses Typus wurden veranstaltet, von denen 14
iiberhaupt keine Nachkommenschaft oder sehr friihzeitige Aborte ergaben.
5 totgeborene Siitze enthielten alles in allem 8 Individuen und 5 lebende Siitze
ergaben 12 Junge. 7 von diesen 12 starben bald nach der Geburt und nur 5
sind am Leben geblieben. 4 der Uberlebenden stammen aus einem Wurf und
das 5. ist das einzig Uberlebende aus einem Wurf von 3 Individuen.

2) Normale Minnchen wurden mit alkoholischen Weibchen gepaart. Das
stellt also die Untersuchung des Einflusses der Mutter dar. In solchen Fiillen
kann der Alkohol die Nachkommenschaft auf zweierlei Weise affizieren: durch
eine Einwirkung auf die miitterlichen Keimzellen oder eine Einwirkung auf den
sich im Uterus entwickelnden Embryo. Nur 4 solche Paarungen gelangten zur
Untersuchung: 1 ergab keine Nachkommenschaft, lebende Siitze wurden 3 ge-
worfen, von denen einer aus 3 friihgeborenen Jungen bestand, welche bei der
Geburt starben. Die andern Sitze bestanden aus je 1 Jungen, das leben blieb.
Die Alkoholbehandlung in einem dieser letzten Fiille setzte erst ein, als die
Mutter bereits seit ungefihr 3 Wochen triichtig war.

3) Alkoholische Minnchen wurden mit alkoholischen Weibchen gepaart.
Das ist der stiirkste Versuch, da ja beide Eltern alkoholisch sind. 14 solche
Paarungen ergaben in 10 Fiillen keine Nachkommenschaft oder sehr friihzeitige
Aborte. 3 tote Wiirfe wurden hervorgebracht mit im ganzen 6 Individuen. und
nur ein einziges lebendes Junge geboren. Dieser einzige Nachkomme
von den 14 Paarungen starb in Konvulsionen am 6. Tage nach der
Geburt.

Die wiihrend der Versuche gestorbenen Jungen zeigten nervise Stérungen,
manche hatten epilepsieihnliche Zufiille und alle starben in Kriimpfen.

9 Kontrollpaarungen von derselben Tiergruppe ergaben 7
lebende Wiirfe, die im ganzen aus 17 Individuen bestanden, die siimtlich
am Leben blieben und fiir ihr Alter kriftige Exemplare darstellen.

42 Paarungen alkoholisierter Meerschweinchen haben nur 18
lebende Junge ergeben, und von diesen lebten nur 7, darunter 5
Kiimmerlinge, linger als einige Wochen, wihrend 9 Kontrollpaa-
rungen 17 Junge ergaben, welche alle am Leben blieben und nor-
male, kraftvolle Individuen sind. Diese Tatsachen demonstrieren
iiberzeugend den schidigenden Einflu8 des Alkohols auf die elter-
lichen Keimzellen und die sich entwickelnde Nachkommenschaft.

(Ubersetzt von W. Gebhardt.

584 Charles R. Stockard and Dorothy M. Craig, An Experimental Study ete.

Literature cited,

BARDEEN, C.R., Abnormal Development of Toad Ova Fertilized by Spermatozoa
Exposed to the Roentgen Rays. Journ. Exp. Zool. Vol. 4. pp. 1—44. 1907.

BertTHoLet, E., Uber Atrophie der Hoden bei chronischem rae
Centralbl. f. allgem. Path. Bd. 20. S. 1062. 1909.

Hertwie, 0., Die Radiumkrankheit tierischer Keimzellen. Bonn, Cohen, 1911.

Hones, C. F., The Influence of Aleohol on Growth and Development. In »Physio-
logical Aspects of the Liquor Problem«. First Ed. by Billings, Houghton,
Mifflin, N. Y. pp. 359—375. 1903.

Marrer et CoMBEMALE, Influence dégénération de l’aleool sur la descendance.
Compt. rend. Acad. de Sc. Vol. 106. p. 667. 1888.

McCuenpon, J. F.. An Attempt towards the Physical Chemistry of the Pro-
duction of One-Eyed Monstrosities. Am. Journ. Physiol. Vol. 29. pp. 289
—297. 1912.

Nicn, L. B., Comparative Studies on the Effects of Alcohol, Nicotine, Tobacco
Smoke and Caffeine on White Mice. I. Effects on Reproduction and Growth.
Journ. Exper. Zool. Vol. 12. pp. 133—152. 1912.

NicLoux, Passage de l’alecool ingéré de la mére au foetus etc. L’Obstétrique.
Vol. 97. 1900.

SrocKarD, C. R., The Influence of External Factors, Chemical and Physical,
on the Development of Fundulus heteroclitus. Journ. Exp. Zool. Vol. 4.
pp. 165—201. 1907.

—— The Artificial Production of a Single Median Cyclopean Eye in the Fish
Embryo by means of Solutions of Magnesium Chlorid. Arch. f. Entw.-
Mech. Bd. 23. 8. 249—258. 1907. Journ. Exp. Zool. Vol. 6. pp. 285—337.
1909.

—— The Influence of Alcohol and Other Anesthetics on Embryonic Develop-
ment. Am. Journ. Anat. Vol. 10. pp. 369—392. 1910.

»Separatabdruck aus dem Zentralblatte fiir Physiologie, Band XXVI, Nr. 7.“

==

Futterungsversuche an Amphibienlarven.
Von J. F. Gudernatseh (New York).

(Vorlaufige Mitteilung.)

(Der Redaktion zugegangen am 9. Mai 1912.)

Es wurde versucht, der zurzeit im Mittelpunkt des Interesses
aller Biologen stehenden Frage, welche Rolle die meisten driisigen
Organe, namentlich die Driisen mit sogenannter ,,innerer Sekretion‘“‘,
im Haushalte des Organismus spielen, auf dem Wege der Verfiitterung
experimentell naher zu treten. Zu den Versuchen wurden im Jahre
-1911 Quappen von Rana temporaria und esculenta verwendet. jetzt
‘werden diese Versuche an den gleichen Tieren und an Bufo- und
Tritonlarven fortgesetzt. Zur Verfiitterung werden Saugetierorgane
verschiedentlichen Ursprungs verwendet, vom Pferd, Rind, Schwein,
Hund, Katze, Kaninchen usw. Es hat sich bis jetzt gezeigt, daB
die Speziesherkunft der Organe ohne wesentliche Bedeutung fiir
ihre Wirkungsweise ist. Verfiittert werden: Thyreoidea, Thymus,
Nebenniere, Hypophyse, Hoden, Ovarium, Milz, Leber, Pankreas
und Muskel. Die genannten Substanzen werden frisch kurz nach
‘Entnahme aus dem Organismus oder, nachdem sie héchstens zwel
Tage auf Eis gelegen sind, verfiittert. Im Jahre 1910 ausgefiihrte
Versuche, den Einflu8 yon Extrakten obiger Substanzen und solcher
verschiedener benigner und maligner Tumoren auf die Entwicklung
‘von Fisch- und Amphibieneiern zu studieren, fiihrten aus AuBeren
Griinden zu keinen befriedigenden Resultaten.

War es a priori zweifelhaft, ob von Sadugern entnommene Organe
bei Verfiitterung an Amphibien auf deren Entwicklung einen spe-
zifischen EinfluB ausiiben kénnten, so hat der Verlauf der Experimente
alle Zweifel dariiber verscheucht. Natiirlich bleibt immer noch die
Frage offen, ob diese Organe, nachdem sie den Amphibiendarm
passiert haben, auf den betreffenden Organismus den gleichen EinfluB
austiben, der ihnen in ihrem Ursprungsorganismus zukommt.

Sehr deutliche Resultate ergeben die Verfiitterungz von Thy-
reoidea und Thymus, zweier Organe, die ja auch bei Wachstum
und Differenzierung des sich entwickelnden Oreanismus eine eroBe
Rolle spielen. Werden sie verfiittert, so sind beide Organe in ihrer
Wirkung auf jene Vorgiinge gerade entgegengesetzt und auch der
Zeitpunkt, auf dem ihr Einflu8 am stirksten in die Erscheinung
tritt, diirfte ein verschiedener sein. Die Thyreoidea scheint am
raschesten zu wirken, je alter die Tiere bei Beginn der Fiitterung
sind, bei der Thymus scheint das Umgekehrte der Fall zu sein,

Wird Thyreoidea auf irgend einem Stadium gefiittert, so hért
jedes Weiterwachstum der Quappen auf und die Tiere schicken
sich sofort zur Metamorphose an. So konnten Quappen, die noch
keine Extremitaten besaBen, innerhalb 7 Tagen dazu gebracht werden,
Hinter- und Vorderbeine zu entwickeln und den Schwanz zu re-
duzieren. In diesem Friihjahr ist es gelungen, Quappen, die erst
16 Tage alt (16 Tage nach dem Verlassen des Eies) waren, zur Bildung
der, Vorderextremititen zu bringen, Da die Thyreoideafiitterung
jedes Weiterwachstum unterdriickt, so sind das Resultat derselben
ganz kleine (Zwerg-) Frésche. Dabei ist es ganz gleichgiiltig, auf welcher
Altersstufe die Behandlung beginnt oder welche Nahrung vorher
verfiittert wurde. Waren die Quappen zu Beginn der Fiitterung
sehr klein, so sind es die metamorphosierenden Tiere auch, gréBere
Quappen aber ergeben gréBere Frésche.

Bei der Verfiitterung von Thymus sind die Resultate gerade
entgegengesetzt. Die Tiere wachsen anfangs sehr rasch, es werden
eroBe Kaulquappen erzeugt, je linger dieselben aber unter dem
Einflusse der Thymus stehen, um so mehr wird die Differenzierung
hinausgeschoben und die Metamorphose eventuell ganz unterdriickt.
So kommt es, daB aus demselben Satz die mit Thyreoidea gefiitterten
Tiere sehr rasch, innerhalb 1 bis 2 Wochen, zur Metamorphose gebracht
werden kénnen, wahrend die mit Thymus gefiitterten Tiere selbst
viele Wochen spater, nachdem die Kontrolltiere schon langst me-
tamorphosiert haben, zum gréRten Teil noch ganz undifferenzierte
Quappen sind ohne Extremitaten und teilweise gar nicht zur Meta-
morphose kommen.

Interessant sind auch Farbungsunterschiede der verschieden
behandelten Tiere, z. B. die tiefdunkle Farbe der Thymusquappen
(Ausbreitung der Pigmentzellen), die auffallend helle Farbe der Neben-
nierequappen (Kontraktion der Pigmentzellen) usw.

Ein eingehender Bericht iiber die Gesamtversuche von 1911
und 1912 sowie iiber die histologischen Ergebnisse und die Beein-
flussung der Regeneration durch die verschiedenen Substanzen wird
spater gegeben werden.

Druck von Rudolf M. Rohrer in Brinn.

ee — Oa ee

~..

Feeding Experiments on Tadpoles.

I. The influence of specific organs given as food on growth
and differentiation.

A contribution to the knowledge of organs with internal secretion.
By
J. F. Gudernatsch,

Department of Anatomy, Cornell University Medical College, New York City.
(From the department of histology, German University of Prague.
Director: Prof. ALFRED Kony }).]

With plate IX.

Eingegangen am 11. Juli 1912.

During a stay in the Zoological Station of Naples, spring 1910,
an attempt was made to study the influence of various organic ex-
tracts on the development of fish (Belone, Gobius etc.) and amphibian
(Rana) eggs. The substances used were extracts from mammalian -
tissues, viz. thyroid, thymus, testicle, ovary, hypophysis, adrenal,
pancreas, cancer of ovary, carcinoma of liver and carcinoma of rectum.
Different quantities of the extracts, in corresponding degrees of con-
centration, were added to the sea-water, containing the recently
fertilized eggs. The eggs were kept for various lengths of time —
up to 20 hours — in this mixture and afterwards transferred into
pure sea-water. In every case an influence upon the developing
eggs was noticeable; and the disturbances of the normal develop-
ment caused by the various substances were different. The difficul-
ties met in these experiments were unusually great, partly on ac-
count of the rapid decomposition of the extracts brought from New

1) My best thanks are due to Prof. Koun for permitting me to work in
his laboratory, furnishing ample material and giving me out of his enormous
experience valuable help and advice as well as for carefully revising the manu-
script.

Archiv f. Entwicklungsmechanik. XXXY. 30

458 J. F. Gudernatsech

York — on board ship they were kept in cold storage, but there
was no ice-box in the laboratory — and partly because in that season
it was very difficult to get sufficient material; in two months Belone
eges were brought to the laboratory only twice. In spite of these
inconveniences a great number of eggs were kept under observation
up to the time of hatching. Yet the work could not be carried on
systematically with sufficient repetitions and control experiments.
Besides it was not to be expected that the influences of the various
organs on development would allow of any conclusions as to the
function of the respective organs, viz. thyroid gland etc. It was
more likely that the disturbances of the normal development were
of a general type caused perhaps by the change in the osmotic
pressure of the surrounding medium ete.

To show that the influences of the various substances upon
development were actually different, the following table may be given.
It cannot, of course, be used for any generalizations; for it is the
result of only one experiment on Belone eggs. The curves show the
respective percentages of the living and developing eggs after a cer-
tain number of days; viz. of each 100 eggs there were on the second
day still living: in the control 87, after addition of ovary exctract 85,
etc. to.... carcinoma recti extract 30; at the beginning of the third
day 76, 65,....26, etc. After the first day (each 100) a different
decline of the curves is already visible. A certain percentage of
the eggs, 13 of the control, died, since naturally not all eggs were
equally able to develop, others were not fertilized ete. For this reason
it may be that on the second day the curves rather run parallel. A
striking decline of all curves with the exception of the control and
thymus curves appears on the fifth day. On this day in normal
development, the heart begins to beat. Many eggs which up to that
time remained alive, although probably with diminished energy, seem
not to have been able to survive this critical point, the starting
of pulsation. From the sixth day on the curves ron more or less
parallel. The control shows after this time a comparatively strong
decline. An explanation for this phenomenon may be that under the
influence of the extracts the more feeble eggs were killed in the
earlier stages of development, while in pure sea-water they were
able to go on developing for some time before their vitality was ex-
hausted.

459

Feeding Experiments on Tadpoles. I.

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T Ode y

460 J. F. Gudernatseh

Later on the advice of Prof. Kony, the various organs have
been applied as fresh feeding material. The tadpoles of Rana
temporaria and Rana esculenta were chosen for the experiments.
These feeding experiments carried on during the summer of 1911
in the histological laboratory in Prague gave some very interesting
results.

The tadpoles were kept in bowls, each containing 15 to 20
individuals, and were fed three or four times a week on the different
organs. Rana temporaria was used in two sets of different ages. As
food were tried: thyroid, liver, adrenal, hypophysis, and muscle
from horse, thymus from calf, testicle and ovary from dog or eat.
Some organs from rabbits and pigs also were given. The origin of
the organs uséd apparently made no difference in their action. The
food was put into the water and was ravenously taken by the ani-
mals. With each experiment one group was left unfed as control
to test how much nourishment the animals could take from the
tap-water which in Prague is very rich on organisms. The water
was changed daily, on hot days sometimes twice. This frequent
change of water as well as the accumulation of the products of meta-
bolism in the water between the changes may have exerted some
influence on the development, yet the prevailing conditions were the
same for all animals. Unfortunately it was found impossible to carry
a constant current of air or water through the great number of dishes.
It was also found impossible on account of the artificial feeding to
keep plants in the dishes. The feeding was continued till shortly
after the appearance of the fore legs, then the animals cease to take
food for some time on account of the transformation of the jaws,
and since breathing through the lungs begins the metamorphosed
animals leave the water and look for fresh food.

Experiment I.

Rana temporaria in two sets of different sizes. Fig. 1a—e,
2a—e. Original size on May 234 1911 of set I 1.1—1.7 em, of
set IL 1.8—2.3 em.

The differences in size of the animals of the same set diminish
somewhat with continued feeding, so that the deviations.from a mean
become less obvious. The following table gives the sizes of the dif-
ferent groups 26 days after the beginning of the experiment:

ee a ee ee ee eee

Feeding Experiments on Tadpoles. I. 461

set I set II

May 23. Original size, average. . . 14 cm 2.05 om
June 18. Average size

control unfed . . . . 2.2 - 3.0 -

ENGaktaij4. Viana, eye: eee 41 -

TARA ci jas} as 9 eee 3.9 -

TUG i605 one beh den ak ay 41 -

LEE MALY ie is (cg 5h te A Lem 3.9 -

hypophysis . ... . 28 - — -

ET ae nee eer As 3.0 -

Ci a aan nti pal Pg 3.3 -

June 14. EpyrOtbnr sis “selieyp2y AR, 18 -

Testis and ovary could not be fed regularly on account of the
material lacking, so that these animals are not much ahead of the
unfed control.

The thyroid fed animals had died on June 16 as fully developed
frogs, though dwarfs in size; therefore their measurement on June 14
is given, on which day the fore legs in set I were also noticeable.

From the notes the following data may be given’):

May 23. Beginning of the experiment.

May 30 (up to this day food had been given 4 times). All thyroid II
show hind legs.

June4. 3 thymus IT show hind legs.

June5. The differences in size between the individual groups become
marked, thyroid I and II are smaller and thymus I and I
bigger than the other animals. The differentiation (limbs,
form of the body etc.) is most evident in thyroid.

June6. More thymus II and some of the other groups grow hind legs.

June?. The differences in size and differentiation have become more
striking. Thymus II are the biggest animals, they have, how-
ever, retained the typical form of tadpoles. Thyroid Il begin
to grow fore legs and to reduce their tails, their bodies are
markedly frog-like.

June8. All thyroid I grow hind legs. Thyroid Il have become typi-
eal, but very small frogs and begin to jump. Some adrenal I

1) For the sake of simplicity, the organ given is used throughout the paper
as an index of the respective group. I and II mean: size I and Il. For in-
stance, liver I means: tadpoles, size I, fed on liver, ete. ‘Thymusthyroid means:
tadpoles, fed first on thymus, later on thyroid.

June 14.

June 16.

June 18.

June 20.

June 23.

June 25.

June 27.

June 29,

July 2.

July 3.

July 8.

July 9.

J. EF. Gudernatseh

and Il show a somewhat lighter color than the rest of the
tadpoles.
Thyroid II begin to die off. Thyroid I grow fore legs and

. start to absorb their tails.

Thyroid I die off. 2 thymus II grow fore legs —i. e. 9 days
later than thyroid If. The lighter coloring of the adrenal I
and II becomes more evident.

Thymus I and II are the biggest tadpoles of each set. The
difference in length between thymus I and II and liver I
and II is not marked, yet the thymus tadpoles are broader,
have stronger legs etc.

The liver show a greenish coloring. Some thymus I and
liver I grow hind legs, i.e. 12 days later than thyroid I.
Thymus II seem to show a retarded differentiation being
behind liver II, muscle If and adrenal IL.

This difference becomes more evident. All adrenal tadpoles
show a color markedly lighter than that of the rest of the
groups.

Thymus I are much bigger, yet far less differentiated than
liver I and musele I.

For several days no more of the thymus II have grown fore
legs, while more and more of the liver II, muscle II, adre-
nal IT and even some of the poorly fed testicle II and ovary II
have done so.

Thymus I and II are very big and their color is very dark. No
more thymus II develop fore legs, while of liver II, muscle II
and adrenal II there is only one in each group without fore
legs. Even of the poorly fed testis II and ovary II there
are only 3, respectively 5 without them. Adrenal I grow hind
legs, muscle I fore legs.

All liver II, muscle II and adrenal II tadpoles have grown
fore legs.

The adrenal II frogs which on July 3 had been taken out of
the water and placed on sand are now just as dark as the
frogs of the other groups. There is only 1 of liver I and 3 of
muscle I without fore legs, while there are still 9 of thymus II
and 10 of thymus I without them, though the latter tadpoles
are much bigger than the former.

Hypophysis I and testis I grow hind legs, adrenal I are the
largest, almost as big as thymus I.

Feeding Experiments on Tadpoles. I. | 463

July 11. Adrenal I grow fore legs.

July 13. There are only 2 of muscle I without fore legs, while there
are still 7 of thymus I, and 5 of thymus I lacking fore legs.

July 17. Some hypophysis I and testis I grow fore legs, the last
muscle I grows fore legs, while there are still 3 of thymus I
and 3 of thymus II without them. Hypophysis I tadpoles
gradually become rather transparent, especially their heads.
They show on the right side of their body a greenish swell-
ing beneath the skin. The green color is also seen through
on the right side of their bodies, yet there is no swelling
there. The tails of some are twisted in a peculiar manner.

July 21. Hypophysis I begin to die one after the other without com-
pleting their metamorphosis. There are still 3 thymus I and
3 thymus II without fore legs, their bodies assume a very
irregular shape and become very broad and bloated.

July 26. The last adrenal I grows fore legs.

Aug.3. 1 more thymus I grows fore legs, there remain 2 without
fore legs.

Aug.4. 1 more thymusI grows fore legs, there remains 1 without
them, 2 thymus II die without fore legs. One only survives.

Aug.5. The last thymus I and thymus II die without fore legs.

Aug. 17. Some of the unfed control grow hind legs.

It is evident from the data just given that the thyroid and thymus
tadpoles (Fig. la—b, 2a—b) reacted most peculiarly to their specific
foods, while the liver, muscle and adrenal animals showed a more
indifferent behavior. However, the very light coloring of the adrenal
was striking as compared with the very dark color of the thymus
and the dark greenish one of the liver tadpoles. It is highly pro-
bable that the light color of the adrenal is not the result of the
feeding with adrenal, but was merely a contraction of the pigment
cells due to the contents of the chromaffine cells going into solution
(adrenalin reaction). The gradually developing transparency of the
hypophysis fed tadpoles must also be mentioned as well as the fact
that most of them died without completing their metamorphosis. The
nature of the green swelling in their abdomen can only be determined
by microscopic examination.

The results of the testis and ovary feeding are inconclusive
since regular feeding was impossible on account of the difficulties
met with in providing the food. The behavior of the other groups,

464 J. F. Gudernatsch

however, is characteristic, and there is also a definite control given,
as the experiments were conducted on two different sets with cor-
responding results.

The quickest results were seen in the thyroid groups. While
an increase in body size was lacking, the differentiation of the body
was extremely rapid, both hind and fore legs appeared earlier than
in any other group and the animals metamorphosed long before those
fed on other substances. It was peculiar that every change in the
body form set in almost simultaneously in all the animals of one set,
so that the corresponding stages of development were reached within
24 hours or less; for instance, all had their hind or fore legs come
out on the same day ete. In no other group could such a uniform
development be-observed. The only explanation of this can be the
increased velocity of the differentiation processes. In the other groups
only a few animals at first began to grow hind or fore legs, and
often many weeks elapsed before all the others had reached the same
stage in development. This is the natural course of events, since
a priori not all tadpoles possessed the same vitality. The thyroid food,
however, enacted such a strong accelerating influence on the body
differentiation that the differences in time which existed in the deve-
lopment of the individual tadpoles were so reduced, that they hardly
remained noticeable. The greatest difference in time, between the
slowest and the most rapid differentiation of thyroid tadpoles of one
set was less than one day.

The difference in time between the thyroid groups and those fed
on other substances was as might be expected greater, the longer
the treatment lasted. For instance, while only 5 days (May 30—
June 4) lie between the appearance of the hind legs in thyroid II
and thymus II, this interval in set I — I is the younger set, therefore
was fed longer — is 12 days (June 8—June 20).

The precocious body differentiation of the thyroid fed tadpoles
did not allow the animals to continue their growth, the result of
the metamorphosis were therefore extremely small (pigmy-) frogs
(Fig. la, 2a).

The feeding with thymus showed an influence on the develop-
ment of the tadpoles, exactly the opposite of that caused by the
thyroid diet. Its consequence was a prolonged increase in size beyond
the normal, the metamorphosis, however, was much retarded or not
completed at all as the animals died before that time. With this
retarded development the individual differences, of course, were much

Feeding Experiments on Tadpoles. I. ; 465

emphasized. Not all individuals of one set grew their hind or fore
limbs on the same day, as in the thyroid groups, but there, were
intervals of days and weeks between the corresponding stages in
different individuals. Those tadpoles that possessed the least amount
of vital energy had to be fed longest. They were, therefore, longest
under the influence of the retarding food.

The later an organ develops in normal ontogeny, for instance
fore legs later than hind ones, the more its appearance was postponed
by the thymus and accelerated by the thyroid diet. The hind legs of
thyroid II and thymus IT appeared at an interval of 5 days (May 30—
June 4), for the fore legs the interval was 9 days (June 7—June 16).
This can also be expressed in the opposite way: the younger a tad-
pole is at the beginning of the feeding, the greater is the retarding
influence on development by the thymus treatment and the accelerating
influence by the thyroid treatment. For instance, in the appearance of
the hind limbs in thyroid II and thymus II (older set) the difference in
time is only 5 days (May 30—June 4), while in thyroid I and thymus I
(younger set) 12 days (June 8—June 20). In this comparison those
thymus fed tadpoles with quickest differentiation, about 10°/), were
chosen. If all the thymus tadpoles were considered, the average dif-
ference in time would be much greater; for thymus I and II, although
fed regularly and abundantly, needed about two months before all
had completed their development, while all the other groups had
metamorphosed long before.

Thus the influence of the thymus food was such that in the beginn-
ing it caused a rapid increase in body size, going beyond the normal,
while later on it postponed the metamorphosis extremely. The color of
the animals became very dark during the experiment. Those tadpoles
most backward in development showed a clumsy bloated shape.

Experiment II.

A group of Rana temporaria tadpoles, originally selected for
ovary feeding, had been fed only twice, May 23 and May 25, with
that substance, after this time, up to the start of experiment II,
July 6, through 43 days, they had starved. The short feeding of
ovary was of so little influence, that these animals differed in no
respect from the unfed control tadpoles (Fig. 3). From July 6 ona
part of these tadpoles were fed on thyroid, another part on thymus.
The differences in the results were most evident (Fig. 6a, 6) and corre-
sponded to those of experiment I.

466

The diary reads as follows:
July 6.
July 9.
July 11,

Start of the experiment.

J. F. Gudernatsch

Average size 2.75 cm.
(after 3 days only) thyroid grow hind legs.
A difference in the sizes of thyroid and thymus is noticeable.

2 thymus grow hind legs.

July 12.

frog-shape.
July 13.
July 14.
July 17.

Size of thyroid 2.6 em, of thymus 3.2 em. Thyroid assume

Thyroid grow fore legs and swim on their back.
Thyroid have completed their metamorphosis and begin to die.
Thymus are very big, entire length 3.7 em (body 1.3 cm, tail

2.4 em) and are very dark colored.

July 18.

2 thymus are still without hind legs. From to-day on these two

will be fed on thyroid, so that the experiment now runs thus:

Thymus.
July 21.

July 22.

July 23.

July 24. Not until to-day 2 of this
group grow fore legs, although
on July 17 they were so much
further along than those in the
right column.

Aug. 5. The last one completes its |

metamorphosis, 22 days later
than thyroid, and 13 days later
than thymusthyroid.

Thymusthyroid.

After 3 days only! Appearance of
hind legs.

Body assumes frog-shape. The
dark (thymus) color has dis-
appeared.

Fore legs! The animals swim on
their back.

Length of body 08em 0.9 cm
= rer Ge eve ae ces 1 es es
entire length 21cm 2.2 cm

Thus experiment II ends with the same results as experiment I.
The feeding on thyroid causes an extremely rapid differentiation of
the body with a complete suppression of growth (compare Fig. 3
and 6a), the feeding on thymus furthers the growth (Fig. 6b), but

retards the differentiation.

This is most strikingly seen in the sub-

Feeding Experiments on Tadpoles. I. 467

experiment described in the right column. Those tadpoles which
were backward most on July 17 metamorphose after having, been
fed on thyroid for only 5 days, sooner than those farthest along in
development, which remained on thymus.

In this experiment the influence of the thyroid food made itself
manifest after only 3 days. The reason for this might be that the
animals had starved through 43 days and had thus become older
without being able to develop. They were, one might say, in a con-
dition of latent overripeness and the first application of food rapidly
caused a further development.

Experiment III.

Taken alone experiment III would not allow of any conclusions,
since it was done with only a few animals. Yet the results attained
are absolutely the same as those of experiments I and IJ, and there-
fore furnish a confirmation of the latter.

Some control animals of experiment I which had been starving
since the first feeding, May 23, through 51 days, were fed on thyroid
from July 13, others on liver.

The experiment ran as follows:

Thyroid. Liver.

July 13. Average size 2.4—2.6 em
July 18. Hind legs appear, tail
gets shorter, body assumes

frog-shape

length of body 0.7 cm 0.9 em
- - tail 0.8 - EO\ =

entire length 1.5 em 2.8 em

July 19. They swim on their back.
July 20. Fore legs just noticeable.

They die off.
July 25. hind legs appear (7 days later
| than in thyroid!).
Aug. 16. | fore legs appear (17 days later

than in thyroid!).

Experiment III again shows the extremely strong influence of
the thyroid food in accelerating the development as compared with
an indifferent food, as liver can be regarded. At the same time it
confirms the above statement, that the differences in time between

468 J. F. Gudernatsch

corresponding stages of development become greater the later an
organ appears in normal ontogeny. The time between the appear-
ance of the hind legs in thyroid and liver was an interval of 7 days,
while the interval between the appearance of the fore legs in the
two sets was 17 days.

To gain a further control of the results of experiments I—III
on Rana temporaria, a similar set of experiments was repeated on
Rana esculenta.

Experiment IV.

Tadpoles of Rana esculenta were fed in groups of 20 on thyroid,
thymus and liver and one group was left unfed. For the feeding on
thyroid 3 groups of different sizes were used, the smallest ones in
group I, the largest ones in III]. Group IL as well as the liver, thymus
and control groups consisted of tadpoles of the intermediate size.

The differences in size at various times of the experiment are
given in the following table:

Control Liver Thymus ey Thyeoid Thyroid

i Ul
July 6. Size at the start of cm cm
the experiment 2.5—3.0 rofl Rimes st S023)
July 17. Length of body!) (106 12 U4) O57 ieee
- - tail 15 , 14). 10) Eee
entire length . 25 | 129, 3A
July 21. Length of body 10° 12 “LA Oi agee aa
- - tail 15 .« Loe 8 yO i ee 6
entire length 25 2.9 - i312) Teen
July 31. Length of body 1.0 | 2a ae
- - tail 1.5.) (ARO zal
entire length 2.5 | SOMRahM
breadth of body — | 06>- "Om
Aug. 10. Length of body 12 15
- = tail 10. ee
entire length | Ba ate
breadth of body O10) Os
- = tail 0.4 0.7

‘) At the beginning of the experiments only the entire length of the ani-
mals was measured. Later it was found better to determine the lengths of the
body and tail separately.

Feeding Experiments on Tadpoles. I. ; 469

Liver Thymus

Aug.16. Length of body 12 + ke
- - tail 2.0: 20
entire length 3.2 4.0
breadth of body 0.72 O08
a b
Aug. 29. Length of body 13 16 1.9 a=smallest
- - tail ra | 2.6 3.2 b= biggest
entire length ae Pe a
breadth of body Oe OF Ev

From the record of the experiment may be mentioned:

July 16. Liver are big and show a greenish color. Thymus are very
big and dark. All thyroid Il and III grow hind legs.

July 17. Thyroid I grow hind legs. Some thyroid IT and III show buds
of fore legs, thyroid II breathe very rapidly and swim on their
backs. The bodies of all thyroid I—III assume frog-shape.

July 20. Thyroid II begin to die, they have typical frog-shape. Thy-
roid II swim on their backs.

July 21. All thyroid II are dead.

July 23. Thyroid I and III begin to die.

July 24. All are dead. Their bodies are typically frog-like.

July 31. The unfed control begin to die. The thymus lose their dark
color and become lighter than liver.

Aug. 13. Liver begin to grow hind legs, 28 days later than thyroid.

Aug 15. Thymus begin to grow hind legs, 30 days later than thyroid.

Aug. 13—Sept.15. Liver die one after the other without completing
their metamorphosis.

Sept. 5—Sept. 7. Thymus die one after the other without completing
their metamorphosis.

This experiment has therefore given the same results as those at-
tained on Rana temp. The effect of the thyroid diet is again striking.

At present it is not clear, why the liver and thymus tadpoles
in spite of the continual feeding (July 6—Sept. 7, Sept. 15) did not
complete their metamorphosis, but died before. The only respect in
which the Rana esculenta experiments differed from those on fempo-
raria was the higher temperature of the water and air. The former
were undertaken during the hottest period of the summer of 1911,
while the latter had been completed before that time. However, it is
unlikely that the rise in the temperature itself should have enacted
such a retarding influence on the development of the tadpoles. One

470 J. F. Gudernatsch

should rather expect the contrary. Although the high temperature
may not be directly injurious, it may indirectly create unfavorable
conditions for artificially rearing the animals. The water in which
the tadpoles were kept contained a large amount of organic sub-
stances constantly undergoing decomposition much more rapidly than
on cooler days. Therefore the accelerating influence of the higher
temperature may well have been counteracted by this process.

Still another reason may account for the delay in development.
Rana esculenta is less fit than temporaria to be reared under arti-
ficial surroundings, therefore in general less resistent to aquarium
conditions. Furthermore, it sometimes happens that under apparently
favorable conditions esculenta tadpoles do not complete their meta-
morphosis before the following spring. BARrurtH and TORNIER state
that overfeeding may postpone the metamorphosis.

The thyroid tadpoles did not succumb to any of the above
mentioned influences. This can easily be explained by the fact that
the thyroid treatment did not have to last very long on account of
the immensely accelerating influence of that food.

During the first half of the experiment the thymus fed animals
showed the same dark pigmentation as the temporaria did, later on this
dark color disappeared and they became even lighter than the liver fed
tadpoles.

Experiment V.

The aim of this experiment was to study the influence that a sud-
den change in the food given would have on the development of Rana
esculenta tadpoles. For this purpose a set of animals which had been
fed on liver since July 6 was on July 21 put on thymus-, another set on
thyroid diet. The same was done with thymus fed animals which were
put on liver and thymus respectively. Thyroid fed tadpoles for feeding
on liver and thymus unfortunately could not be used. With other ani-
mals it was tried, however, to stop the rapid progress in differentiation
after thyroid diet by giving liver or thymus, but without results.

a. Liver fed tadpoles, put on thymus or thyroid
diet on July 21.

Liverthymus. Liverthyroid.
Average size:
July 21. Length of body 1.2 cm 1.2 cm
= jyg tail. )1.t0- EA ee

entire length 2.9 cm 2.9 cm

Feeding Experiments on Tadpoles. I. 471

Liverthymus. Liverthyroid.

July 24. After 3 days feeding! hind, legs
. appear.
July 27. Frog-shape is noticeable.
Length of body 1.2 cm 11 ene
- - tail 21 - 16 -
entire length 3.3 em 2.7 em
July 31. Length of body 1.3 em 1.0 em
- - tail 23 - 14 -
entire length 3.6 cm 2.4 em
breadth of body 0.75 - 0.6 -, length of legs 0.3 em.
Swim on the back, air vesicles in
(continued unter c.) the gill region, begin to die off.

b. Thymus fed tadpoles, put on liver or thyroid
diet on July 21.

Thymusliver. Thymusthyroid.
July 21. Length of body 1.4 cm 1.4 em
ey Sj atall. » 1.5) "= 1.8 -
entire length 3.2 cm 3.2 em
July 24, After 3 days feeding! hind legs
appear.
July 27. Frog-shape is noticeable.
Length of body 1.4 cm 1.3 em
puesta 2. 1.8 -
entire length 3.5 cm ~—83.1.em |
July 30. Length of body 1.4 cm 1.1 em
= ol ee 15 -
entire length 3.5 c¢m 2.6 em
breadth of body 0.7 - 0,6 -, length of legs 0.6 em.

Swim on the back, air vesicles in
the gill region.
Aug. 2. Begin to die. A few have the buds
of the fore legs out.
Aug. 3. The last ones die.
Length of body 1.0 em
- -tail 15 -
entire length 2.5 em
breadth 0.6 -
(continued under c.) length of legs 0.6 -

472 J. F. Gudernatsch

c. Continuation of the left columns of the above tables.
A comparison of liverthymus and thymusliver.

Liverthymus. Thymusliver.

July 24. Length of body 1.2 em 1.4 em
=) (= fail dee 18 -

entire length 2.9 em 3.2 cm

July 27. Length of body 1.2 em 1.4 em
- -ftail 21 - 2.13

entire length 3.3 cm 3.5 em

July 31. Length of body 1.3 cm 1.4 em
=. = tally 23 2.2 -

entire length 3.6 cm 3.6 em

breadth of body 0.75 - 0.7 -

Aug.5. Length of body 1.45 cm 1.4 em
- -tail 25 - 2.3 -

entire length 3.95 cm 3.7 em

breadth of body 0.8 - 0.7 -

Aug. 10. Length of body 1.5 em 1.4 cm
“= tail 216 a 2.3 -

entire length 4.1 cm 3.7 em

breadth of body 0.8 - 0.7 -

Aug. 13. Hind legs appear, 20 days later than in thyroid (compare
the right columns of a and Db).

Aug. 16. Length of body 1.6 em 1.4 cm
- - tail 28 - 2.3 -
entire length 4.4 em 3.7 cm
breadth of body 0.8 - 0.7 -
Sept. 2. Begin to die without developing
fore legs.

Sept. 20. Begin to die without de-
veloping fore legs.

Experiment V shows that a thyroid diet, started even at an ad-
vanced stage of differentiation and after other substances have been
fed, is able to influence the further development intensely. It seems
of little importance, which substances were fed before the thyroid, ex-
cept that the relative sizes are different. The liverthyroid went almost
parallel with the thymusthyroid (Fig. 8a, b). Some minute differences,
however, were noticeable, yet further experiments with a combined

Feeding Experiments on Tadpoles. I. 473

diet will have to determine their importance. Some of the thymus-
thyroid, for instance, showed buds of the fore legs, while the liver-
thyroid died before that stage showing characteristic responses to
thyroid. These features as swimming on the back, formation of air
bubbles in the gill region and others will be discussed later.

The liverthyroid and thymusthyroid were far ahead of the liver-
thymus and thymusliver and also of the liver and thymus of experi-
ment IV.

The liverthymus and the thymusliver ran almost parallel with
the exception that the liverthymus grew bigger than the thymus-
liver. The thymus diet, therefore, furthers growth even, when it is
given at an advanced stage of differentiation, but apparently less than
when given to younger animals.

The following comparison of tables IV and V is interesting:
liverthymus Ve become gradually larger than liver IV, thymusliver Ve
smaller than thymus IV. The thymus food thus seems to act differ-
ently at different ages, and it may be possible to find a time or stage
for its optimum influence such as is also surmised for the thyroid diet.

In liver IV the hind legs appeared on August 13, in thymus IV
on August 15. This difference in time is rather small and further
experiments must show, whether or not it is significant. At any rate,
this observation agrees with those made on Rana temporaria, which
showed that the thymus food retarded the development. Liverthymus V
and thymusliver V grew their hind legs on August 13, i. e. on the same
day as liver IV. Thus the partial feeding on thymus seems not to
have caused the same delay in development as the exclusive thymus
diet. However, a difference of only 2 days, observed on one set of
animals does not allow of conclusions.

Experiment VI.

This experiment can be regarded as a supplement to experiment V,
at the same time it furnishes a further confirmation of the results of
former thyroid feedings. Tadpoles that had been fed on liver and
thymus 15 days longer than the corresponding groups of experiment V,
thus were 15 days older, were put on the thyroid diet on August 5.

This last experiment shows that the thyroid when food given even
at a very advanced stage of differentiation can cause an accelerated
development. 5 days after the beginning of the experiment hind legs
appear, this is still 3 and 5 days sooner than in the control animals
liver IV and thymus IV. The effect of the previous feeding on different

) Archiv f. Entwicklungsmechanik. XXXY. 31

474 J. F. Gudernatsch

Liverthyroid. Thymusthyroid.
Aug. 5. Length of body 1.2 cm 1.4 em
=)eyitss dail, ci. Dus 2.2 -
entire length | a8 hem 3.6 em
breadth of body 0.6 - 0.8 -
Aug. 10. Length of body 1.2 em 1.3 em
- — tail 18 o- Dalat
breadth of body 0.6 em 0.7 cm
Hind legs appear after 5 days feeding. (The liver IV and thy-
mus IV do not grow them until August 13 and August 15.)
Aug. 12. Frog-shape is noticeable.

Aug. 14. Swim on the back.

Aug. 15. 2 grow fore legs.

Aug. 16. These two (a, b) die, the
rest grow fore legs

a b rest

length of body 0.9 1.05 0.9 cm 1.15 em
- - tail 1210 12 - a
entire length 2.1 2.05 2.1 cm 2.85 cm
breadth of body | 0.5 em 0.7 -
Aug. 17. Begin to die off. Swim on the back.
Aug. 18. 1 grows fore legs.
Aug. 19. Last ones die Last ones die.
smallest largest smallest largest
length of body 0.8cem 0,9 cm 1.0 em 1.1 em
- = -tail), 0'6>— 039 = 1.2 - 14 -
entire length 14cm 1.8cm 2.2 cm 2.5 cm
breadth of body 0.5 - 0.5 - 0.65 - 0.7 -

substances before the thyroid diet here also manifests itself in the
different sizes of the animals. During the entire experiment thymus-
thyroid remain bigger than liverthyroid; on the other hand liverthyroid
develop quicker than thymusthyroid, which is suggested also by ex-
periment V. If further experiments of this kind give similar results,
we shall have additional evidence, that thymus food postpones the
metamorphosis. In fact, at the beginning of experiment VI the liver IV
must have been ahead of thymus IV, although macroscopically the
difference was not evident; for liver IV grew their hind limbs on Au-
gust 13 and thymus IV on August 15.

Feeding Experiments on Tadpoles. I. 475

General discussion.

The most striking and at the same time unquestionable results
were attained by thyroid feeding. They were the same in all ex-
periments. The influence of the thyroid food was such that it stopped
any further growth but on the contrary led to an abnormal diminution
of the size in the animals treated, while simultaneously it accelerated
the differentiation of the body immensely and brought it to a pre-
mature end. It was of little importance, at which stage of differen-
tiation the thyroid diet began or which kind of food had been given
before. Under all circumstances the influence of the thyroid food
became noticeable in a very short time.

This influence must have been very strong, as can be concluded
from two kinds of observations. First, within a very short time, 3—d
days, after the beginning of the experiments changes in the outer
features of the animals were noticeable; second, the influence on all
tadpoles of one group was uniform and rather parallel. While, for
instance, in other groups not fed on thyroid the influence of the food
became evident gradually, without abolishing the individual differences,
so that the individuals of one group grew their hind legs, fore legs ete.
one after the other, often at intervals of many days, the thyroid diet,
on the other hand, brought all the animals of one group within a
few hours, not more than 24, to the same stage of development.
However, it cannot be said that the individual differences were entirely
abolished. The measurable signs of these differences, the intervals
between the corresponding phases of development, were greatly reduced
since the entire period of development was much shortened.

One of the most peculiar features is that the time at which the
feeding begins is of no importance as regards its results. The stages
of development of the animals to be treated may be chosen, but al-
ways the same results will be obtained. Animals in different stages
of development, others that had starved for many weeks, and still
others that had before been fed on other substances were placed on
thyroid diet with exactly the same results: within a few days the
rapid differentiation of the body began. Thus extremely young or
very old tadpoles could be forced to undergo their methamorphosis
quickly. The lower and upper limit of age for the start of a sue-
cessful thyroid diet will be determined later. The upper limit is pro-
bably the time shortly before completing their metamorphosis, when
the tadpoles stop feeding in general. How near to the time of hatching

31*

A76 J. F. Gudernatsch

the lower limit can be brought further experiments will show!). The
tadpoles that were available for the experiments here recorded had
been hatched for some weeks.

The second influence of the thyroid diet, the suppression of growth,
is merely the consequence of the precocious development, and this in
turn seems to be caused by the well known activity of the thyroid
agens to stimulate metabolism. The thyroid agens accelerates the
metabolism which leads to a rapid reduction of the larval organs and
thus to a premature metamorphosis. As soon as thyroid food is given
the differentiation of the body begins. Hand in hand with the progress-
ing metamorphosis goes, more than in the case in normal development, a
reduction of the body mass (resorption of the tail, loss of water, there-
fore an increasing compactness of the body etc.) The outcome of such
precocious metamorphosises are then very small (pigmy) frogs. This
mass reduction was especially striking in the experiments on Rana
esculenta. 7

The thyroid showed still other peculiar influences on the behavior
of the tadpoles. Towards the end of the metamorphosis the animals
hardly moved about in the water. They were always lying quietly,
generally on their backs. When disturbed they would move for a
few seconds in a somewhat convulsive manner and then drop again
to the bottom of the dish, while tadpoles fed on other material
would swim about for a long time. The reason for this may be that
the thyroid fed tadpoles always began to reduce their tail before the
extremities were at all or sufficiently strongly developed. The ex-
tremities, even if fully developed, were always extremely thin, merely
thread-like (Fig. 6a), and could hardly be used for swimming a long
time.

At one time Rana esculenta tadpoles of the different groups were
placed in small dishes with equal quantities of water, to which equal
amounts (about 5 drops) of chloroform had been added. This was
done so as to be able to photograph the animals. All tadpoles remained
the same length of time in the mixture. All animals survived the
narcosis very well except the thyroid fed ones which died in it.

At another time Rana temporaria tadpoles were taken out of
the water and placed on wet filter paper to photograph them. Dur-
ing this procedure, which of course was somewhat rough, the thyroid

‘) In recent experiments (1912) which will be discussed in a later paper

I succeeded in forcing Rana temporaria tadpoles to grow fore legs as early
as 15(!) days after leaving the egg.

Feeding Experiments on Tadpoles. I. 477

died, while the others stood it. Thus in different ways it was seen
that the thyroid fed tadpoles possessed far less resistance against
noxious influences than the others, as if the thyroid food had weakened
their systems enormously. One cannot, however, speak of a poisoning
of their body in tle true sense, since that would not have allowed
the rapid progress in development.

In the tables given above several dates are mentioned at which
the animals began to swim on their backs. This, too, is one of the
features observed only in thyroid tadpoles. Before the animals com-
pleted their metamorphosis, about 3—4 days previous, they began
lying on their backs and floated passively on the surface of the water.
They breathed very heayily and rapidly. Even when disturbed and
swimming actively they did not usually turn over. It seemed as if
the animals were passively forced to take this peculiar position;
as under the skin in the gill region there were always one or two
air bubbles visible, as if during the closure of the gill opening air
had been enclosed. If the animals did not die these air bubbles
were usually absorbed after which the animals assumed a normal
position. It was seen that the swimming on the back always began
shortly before the completion of the metamorphosis and its early ap-
pearance was watched.

The influence of the thymus diet on the development of the
tadpoles was as evident as that of the thyroid, but less striking,
The thymus food caused an accelerated growth beyond the normal
(giant tadpoles) and at the same time it retarded or completely sup-
pressed the differentiation of the body. In doing so individual differ-
ences were very much emphasized, so that an interval of several
weeks elapsed between the metamorphosis of the first and the last
tadpole, while in normal development the difference amounted to
days only. The strongest tadpoles or better those which at the start
of the feeding had progressed most in their development were best
able to keep pace with the control. Those, however, which were back-
ward in their development at the time the thymus diet began stayed
much behind the control, since they were attacked by the thymus
at a less advanced stage of differentiation, and further because they
remained longest on thymus diet.

The thyroid and thymus diets were thus diametrically opposite
in their influences. Their relative action, however, corresponds with
the views held regarding the physiological properties of these organs.

478 J. F. Gudernatsch

Experiments of the kind discussed in this paper may perhaps give
a direction for further studies towards a rational application of thymus
and thyroid preparations.

It is not the purpose of this experimental paper to discuss the
extensive literature on the functional and therapeutic importance of
the organs with an internal secretion. Reference is simply made to
the numerous papers in which the therapeutic value of thyroid pre-
parations for the stimulation of metabolism and ossification, and the
influence of the thymus on growth in the early periods of individual
life are being discussed. A list of them will be found at the end of
this paper.

Liver and muscle were about equal in their action on devel-
opment and did not seem to influence especially the normal progress
of differentiation. Since so far they appeared to be indifferent food
stuffs the tadpoles fed on liver or muscle were regarded as a control
to the other feedings. However, under natural conditions the animals
have a food supply quite different from a constant meat diet, yet
for various reasons it was impossible to study the development of
control animals on a more vegetal or mixed diet!).

The tadpoles fed on adrenal?) developed somewhat slower than
those fed on liver or muscle, otherwise quite normally. The outcome
of the metamorphosises were especially large and strongly developed
frogs (Fig. 55). Sic

A prolonged diet of hypophysis did not force the animals to
complete their methamorphosis. They all died before that stage. No
conclusions can be drawn from this fact, since these tadpoles were
not fed as regularly as the others on account of great difficulties
encountered in providing the food’). The feeding on testis and ovary
was also unsatisfactory for the same reason.

') Such experiments are now being done, spring 1912, and they will be dis-
cussed in a later paper. The difference in macroscopic development between
a vegetal and liver or muscle diet is slight.

*) Experiments on feeding adrenal cortex and medulla separately will be
discussed in a later paper.

3) More extensive experiments on feeding the two lobes of the hypophysis

will be discussed in a later paper. So far they do not confirm the above
statement.

Feeding Experiments on Tadpoles. I. 479

Preliminary experiments were also undertaken on Rana escu-
lenta to study the influence of different diets on regenerating
animals. So much can be said that of tadpoles which hat a piece
of their tail amputated the thymus fed ones regenerated quickest,
while the thyroid fed ones, although they did regenerate a part,
showed the typical precocious metamorphosis. In one experiment the
average length of the regenerated part of the tail was: in thymus
3.9 mm, in thyroid 3.2 mm, in liver 2.9 mm; in another experiment:
liver 3.1 mm, thymus 4.6 mm; later liver 6 mm, thymus 9mm. Re-
generation of the tail begins even when the animals are near the
point where they resorb their larval organs, otherwise the thyroid
fed ones would not have regenerated. BarrurrH showed that Rana
fusca tadpoles which metamorphosed even 2 or 3 days after the
operation tried to regenerate the amputated part of their tails.

The influence of the different food stuffs on the pigmentation
has been mentioned before. The animals were kept under the same
conditions of light and temperature and in the same kind of dishes.
The position of the dishes was changed daily in a certain rotation so
that the minute differences in light and temperature were abolished
as much as possible.

The liver fed tadpoles were rather dark, gradually assuming a
greenish tint. The thymus fed tadpoles of Rana temporaria grew
extremely dark with the progress of the experiments until they be-
came almost black; those of LR. esculenta grew dark in the beginning,
later, however, they became lighter. The adrenal fed tadpoles after
3—4 weeks became extremely light in color. Those fed on hypophysis
lost their pigment more and more and became almost transparent, but
this may have been the consequence of the irregular feeding.

Tornier has studied the influence of varying quantities of food
on the pigmentation of Pelobates fuscus tadpoles and found that a
minimum food ratio gives albinotic, a maximum ratio highly melanotic
larvae and frogs. So the melanism of the thymus fed tadpoles may
have been partly caused by an overrich diet, yet they were much
darker than those in the other groups, although all were fed suf-
ficiently well. Why the Rana esculenta larvae which in the first
weeks of the experiments were as melanotic as the temporaria, later
lost their dark appearance, cannot be explained at present. Very
minute differences in temperature, as KAMMERER points out, may
easily cause a change in pigmentation.

480 J. F. Gudernatsch

The very dark and the very light adrenal (cortex and medulla)
tadpoles seem, roughly estimated, to have possessed equal amounts
of pigment. In the thymus fed animals the pigment cells were spread
out very much in a star-like manner, in the adrenal fed ones they were
completely contracted. Former experiments with adrenalin would
warrant the suggestion that the extract from the chromaffine cells of
the medulla which dissolved in the water caused the pigment cells
to contract‘).

The histogenetic processes must have been influenced very
much by the different diets. The investigation of the thyroid and
thymus fed material promises especially interesting results. The re-
port on this topic will be given later.

More experiments, especially with mixed diets, are necessary
to clear up all the questions concerned in this discussion. At any
rate, these experiments may open a new and extensive field of work
in experimental morphology, in which success is rather certain.

At present one fact alone deserves notice, that the food stuffs
given fresh were able to pass the stomach without losing at least
some of their specific properties. It still remains an open question,
whether their action, after they have passed the intestinal canal, is
_ entirely the same as that which they exert as functionating organs.
Before this question is solved, no conclusions can be made on the
role of these organs in the household of the body. However, so far
it has been shown that a diet on thyroid substance or the applica-
tion of thyroid tablets can to a certain degree substitute the normal
function of the thyroid gland. — It must also be kept in mind that
mammalian organs were fed to amphibians.

Summary.

A number of mammalian organs, especially those with an internal
secretion, thyroid, thymus, adrenal, testis, ovary, hypophysis, liver,
muscle ete. were given as food to tadpoles of Rana temporaria and
esculenta. It was seen that each organ exerted a certain influence
on growth and differentiation of the animals. Most striking was the

‘) Compare: LIEBEN, 8., list of literature. Recent (1912) experiments, how-
ever, so far indicate that the feeding on adrenal cortex causes a much lighter
pigmentation than an adrenal medulla diet.

Feeding Experiments on Tadpoles. I. 481

influence of the thyroid food. It caused a precocious differentiation
of the body, but suppressed further growth. The tadpoles began to
metamorphose a few days after the first application of the thyroid
and weeks before the control animals did so. The influence of the
thymus was quite the opposite, especially during the first days of
its application it caused a rapid growth of the animals, but postponed
the final metamorphosis or suppressed it completely. The action of
the other organs must be studied further before definite statements
can be made. The thymus diet gave very dark, melanotic tadpoles,
the adrenal diet extremely light albinos, the liver diet dark ones
with a greenish tint.

Zusammenfassung,

Verschiedene Siiugetierorgane, namentlich solche mit innerer Secretion,
Thyreoidea, Thymus, Nebenniere, Hoden, Eierstock, Hypophyse, Leber, Muskel
usw. wurden an Kaulquappen von Rana temporaria und esculenta vertiittert. Jede
Fiitterung iibte einen andern EinfluB auf das Wachstum und die Differenzierung
der Tiere aus. AuGerst auffallend war die Wirkung der Schilddriisennahrung.
Sie verursachte eine rapide Kérperdifferenzierung, die zu einer vorzeitigen Meta-
morphose fiihrte, wobei aber jedes Weiterwachstum unterdriickt wurde. Die
Kaulquappen begannen ihre Metamorphose wenige Tage nach der ersten Schild-
driisendosis und um Wochen friiher als die Kontrolltiere. Der Einflu8 der
Thymusnahrung war gerade entgegengesetzt. Sie bewirkte namentlich in den
ersten Tagen ein schnelles Wachstum der behandelten Tiere, schob aber die
Metamorphose immer weiter hinaus oder unterdriickte sie giinzlich. Der EinfluB
der iibrigen Organe mu8 noch weiter studiert werden. Die Thymusverfiitterung
ergab tief dunkel, fast schwarz gefiirbte Quappen, die Nebenniere ganz lichite,
albinotische Tiere, die Leber dunkle, mit einem Stich ins Griinliche.

List of Literature.

BarFurtTH, D., Versuche iiber die Verwandlung von Froschlarven. Arch. f.
mikr. Anat. 1887. Bd. 29. S.1.

— Der Hunger als firderndes Prinzip in der Natur. Arch. f. mikr. Anat.
1887. Bd. 29. S. 28.

Bascu, K., Beitriige zur Physiologie und Pathologie der Thymus. Jahrb. f.
Kinderheilk. 1906. Bd. 64. 1908. Bd. 68.

Brep., A., Innere Secretion. Wien-Berlin 1910.

GuDERNATSCH, J. F., liitterungsversuche an Kaulquappen. Demonstr. Verh.
Anat. Ges. 26. Vers. Miinchen 1912.

—— Fiitterungsversuche an Kaulquappen. Vorl. Mitteil. Centralbl. f Physiol.
1912.

Hammar, I. A., Fiinfzig Jahre Thymusforschung. Ergebn. d. Anat. u. Entwick-
lungsgesch. 1910. Bd. 19.

482 J. F. Gudernatsch

KAMMERER, P., Kiinstlicher Melanismus bei Eidechsen. Centralbl. f. Physiol.
1906. Bd. 20. S. 261.

—— Uber kiinstliche Tiernigrinos. Verhandl. zool.-bot. Gesellsch. Wien. 1907.
Bd. 57. S. 136.

—— Die Wirkung iiuBerer Lebensbedingungen auf die organische Variation im
Lichte der experimentellen Morphologie. Arch. f. Entw.-Mech. 1910.
Bd. 30. S. 379.

Lipsen, S., Uber die Wirkung von Extrakten chromaffinen Gewebes (Adrenalin)
auf die Pigmentzellen. Centralbl. f. Physiol. Bd. 20.

TornieR, G., Nachweis iiber das Entstehen von Aibinismus, Melanismus und
Neotenie bei Fréschen. Zool. Anz. 1907. Bd. 32. S. 284.

VINCENT, SWALE, Internal Secretion and the Ductless Glands. Lancet. 1907.

Explanation of Figures,

Plate IX.

Fig. la—e. Rana temporaria set I (smaller size), photographed June 11 1911.
Natural size. a@ tadpoles fed on thyroid, already changing into frogs. Tail
is shortening, fore legs appear. 6 tadpoles fed on thymus, ¢ on liver,
d on muscle, e on adrenal.

hig. 2a—e. . Rana temporaria set II (larger size), photographed June 11 1911.
Natural size. a—e as in Fig.1. The thyroid fed tadpoles have all meta-
morphosed, the tail in some has almost disappeared, fore legs are well
developed.

Fig. 3. Rana temporaria tadpoles that had been used as an unfed control in
experiment I, thus starved till July 13. Photogr. July 13. Nat. size.

Fig. 4. The same tadpoles as in Fig. 3, photogr. 7 days later, July 20. a@ had
been fed in the mean-time on thyroid, and are already metamorphosing
into pigmy frogs. 0b had been fed on liver. These tadpoles do not meta-
morphose until 19 days later.

Fig. 5. Rana temporaria set I frogs. Photogr. July 25. Natural size. a fed
from the beginning of experiment I on adrenal. 6 one of the tadpoles
that originally had starved. Their size on July 6 was that of the tadpoles
in Fig. 3. From July 6 to July 17 these tadpoles, 54, were fed on thymus,
from July 18 on thyroid. On July 21 hind legs appeared, July 23 fore legs.
Notice the small body and the much shortened tail of a frog metamor-
phosing under thyroid influence, while the adrenal frogs, 5a, at the time
of metamorphosis are large and still have their long tadpole tails.

Fig. 6. Eana temporaria set I, photogr. July 13 1911. Natural size. Animals
that originally had starved. Their size on July 6 was that of the tadpoles
in Fig. 3. From July 6 to July 13. @ were fed on thyroid, b on thymus. a are
changing into pigmy frogs, fore legs appear, tail shortens, } are still huge
tadpoles. Compare also these thymus tadpoles with Fig. 46 fed for the
Same time on liver.

The animals in Fig. 4—6 are all of the same age and the same original
size, set I, but fed on different organs.

lig. 7. Rana esculenta, photogy. August 9, nat. size. a fed on thymus since July 6,

b fed on liver since July 6, ¢ on thymus from July 6 to July 20, on liver

since July 21, d fed on liver from July 6 to July 20, on thymus since July 21.

Feeding Experiments on Tadpoles. I. 483

a and c, d which either entirely, a, or at a time of their development,
c, d, were fed on thymus, are much bigger than those fed on liver only, d.
Fig. 8. Rana esculenta, photogr. August 19 1911, nat. size. The animals were
dead, when being photographed, therefore the curved tails. @ fed from
July 6 to August 5 on thymus, from August 6 to August 18 on thyroid. 6 fed
from July 6 to August 5 on liver, from August 6 to August 16 on thyroid.
The animals in Fig. 7 and 8 are all of the same age, but fed on dif-

ferent substances, and were photographed on the same day.

Anmerkung. Vorliegende Untersuchungen wurden im Sommer 1911 in
meinem Institute durchgefiihrt. Das Manuskript war bereits im Dezember 1911
druckfertig. Eine schwere Krankheit, die mich lange zur Untitigkeit zwang,
hat die Veréffentlichung verzigert. .

Prag, Mai 1912. Prof. ALFRED Koun.

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are

Reprinted from THE AMERICAN JOURNAL OF ANATOMY VOL. 13, No. 2
May 1912

THE BEHAVIOR AND RELATIONS OF LIVING CON-
NECTIVE TISSUE CELLS IN? THE FINS OF FISH
EMBRYOS WITH SPECIAL REFERENCE TO THE
HISTOGENESIS OF THE COLLAGINOUS OR WHITE
FIBERS

JEREMIAB S. FERGUSON

Assistant Professor of Histology
Cornell University Medical College, New York City

TEN FIGURES

In the process of connective tissue development the cells first
arise, the fibers later appear. This sequence is established be-
yond controversy. The ontogenetic relation of cell and fiber is
not, however, so thoroughly established. The theories advanced
may be grouped under three heads: (1) Intra-cellular origin, the
cells may transform into fibers (Schwann, Valentin, Boll, Flem-
ming, Spuler, Livini). (2) Extra-cellular origin, the fibers arise in
the intercellular substance by its fibrillation, or possibly as a
secretion from the cells (Henle, Merkel, Virchow, Kédlliker).
(3) Epicellular origin, the fibers form in an ectoplasm at the sur-
face of the cell (Schultze, Hansen, Golowinski, Mall).

These theories have all been primarily founded upon the results
of examination of ‘fixed’ or ‘killed’ tissue, or upon the study of
fresh teased tissue. Living connective tissue has been studied in
the mesentery of the frog and other animals under conditions
which are accompanied by marked inflammatory reaction and
certain stages of the formation of exudates and of scar tissue
have been thus investigated, and more recently movements of
connective tissue cells have been observed in tissue cultures but
so far as I know the theories of the histogenesis of connective
tissue have not been examined with reference to the behavior of
living cells under normal conditions.

129

130 JEREMIAH S. FERGUSON

Living connective tissue cells have been seen in tissue cultures
by Harrison, Burrows, Carrel and Burrows, Margaret R. and W.
H. Lewis and others, to exhibit a certain motility, and Harrison
has recently emphasized the stereotropic tendency of connective
tissue cells in cultures when in contact with foreign surfaces,
glass, spider-web, etc. But so far as I know, the histogenesis of
connective tissue fibers has not been so studied, and at best the
culture method is open to some criticism on the ground that while
the connective tissue cells are undoubtedly alive and active, yet
they exist under very unusual, if not abnormal, conditions whose
effects have not yet been subjected to complete analysis. Under
these conditions the behavior of the connective tissue elements
while probably similar, is not certainly in exact conformity with
that of the tissue within the embryo.

In order that deductions based upon these several methods of
examination be adequately controlled it appeared desirable that
developing connective tissue be studied in the living animal under
conditions which were in every respect normal, or which, at least,
resulted in no inflammatory reaction. In mammals this endeavor
is fraught with considerable difficulty owing to the size of the
mammalian embryo and the depth beneath other tissues, often
not transparent, at which the connective tissue lies.

During the past summer I had the opportunity, through the
courtesy of the Marine Biological Laboratory at Woods Hole, of
studying connective tissue in the fins of living fish embryos under
conditions which were wholly normal and unaccompanied by
any evidence of inflammatory reaction.

If a free swimming Fundulus embryo is placed on a hollaw
ground shde it will continue to swim, often actively, and its heart
beat and circulation are maintained. It may be observed for
some minutes and at the end of observation may be returned to
the aquarium to continue an uneventful existence for hours or
days thereafter. If a drop of chlorotone is added, or frequently
without its addition, the fish will remain quiet for some minutes,
thus permitting continued observation of connective tissue cells
in his semitransparent fins. Certainly cells studied under these
conditions are open to no criticism of abnormality.

’

LIVING CONNECTIVE TISSUE CELLS 131

The viability of the animals is unaltered for I have kept them
for several days after such observation without any indication of
decreased activity on their part. Even embryos which have been
quieted by chlorotone, as well as those immersed for hours in a
solution of Bismark brown in sea water, I have resuscitated and
kept alive and in an apparently normal and usual condition for
two or three days; they could easily have been kept longer had it
seemed advisable.

The tissue selected for observation was in the fins of free swim-
ming pelagic and Fundulus embryos. The embryos studied were
chiefly of Fundulus and varied from 5 to 20 mm. in total length.
The most favorable subjects were from the time of hatching, 5
to 6 mm., up to 12 mm. in length. The pectoral and caudal fins
were usually selected as most available for observation. In
such embryos the fin consists of a central frame work formed by
the jointed rays, lepidotrichia, with their attached muscles, and
a superficial integument of pavement epithelium with its sub-
jacent basement membrane. The finer fin rays, actinotrichia,
continue the jointed rays to the margin of the fin. The fin at
this stage is very thin and the epidermis lies almost in contact
with the finrays. But between adjacent rays is an interval which
lodges on either side the afferent and efferent blood vessels, bor-
dered by chromatophores, and between them a loose mass of mes-
enchymal connective tissue in which the cells may be readily
observed.

In embryos 5 to 6 mm. long the connective tissue in the pectoral
fins consists chiefly of a mass of round cells confined to the proxi-
mal portion, and beyond this mass a distal fringe or ‘skirmish
line’ of scattered stellate cells. In the unpaired fins, which are
less advanced in their development only the scattered stellate
cells are represented, the invasion of the round cell mass having
not yet occurred. In later stages, as in the caudal fin of the same
embryo, the zone of round cells has advanced distalward among
the actinotrichia nearly to the fin margin, leaving behind between
the lepidotrichia an area of more mature cells, stellate and spindle,
and a few fine fibers well separated by broad spaces occupied by
tissue fluids. The spindle cells and fibers preponderate in the

132 JEREMIAH S. FERGUSON

proximal, the round cells in the distal zone of the fin. Hence,
one follows the sequence of development in passing from the dis-
tal toward the proximal portion of such a fin. Older embryos
show the same zones of transition but in them the formation of
fibers in the proximal region is more advanced.

In mammalian tissue one finds three stages in the histogenesis
of connective tissue, a primitive cellular stage, a syncytial stage,
and a fibrous stage. The first is characterized by the predomi-
nance of round cells, the second by stellate, the third by spindle
and lamellar cells. 'The same succession of cell types is present
in the finsof embryo fish and there is a corresponding succession of
histogenic stages. Fibers do not appear prior to the appearance
of cellular processes. Fine fibers appear coincidently with stel-
late cells, coarsé fibers and fiber bundles develop later.

In the distal portion of the fin fine fibers first appear in the round
cell area coincidently with the transition from round to early
stellate forms. At exactly this period I have observed the first
indication of motion, the throwing out of pseudopods by the round
cells, in the connective tissue cells of the living embryo. Fig. 1
shows such changes in two cells on the border of the round cell
area near the posterior end of the ventral fin. There is at this
time relatively little locomotion, as is shown in the figure by
referring the position of the cells a and 6 to the relatively fixed
point, a prominence on the margin of an adjacent chromatophore
(ch).

The first appearance of fibers in the distal portions of the fins
has been very properly connected by Harrison, and by Goodrich
with the origin of the dermal fin rays from the ‘scleroblast’ cells
which closely resemble the connective tissue cells and like them
are of mesodermal origin. In the region of the actinotrichia
in the distal portion of the fin, it is difficult to distinguish between
the early forms of these coarse fibers and the true connective tis-
sue fibers, but the actinotrichia are confined to the region of the
last one or two joints of the jointed fin rays, and there they pro-
ject, as Goodrich has shown, from between the two opposed
dermal plates which form the distal section of the jointed fin ray.
If therefore one studies a region proximal to the last section and

LIVING CONNECTIVE TISSUE CELLS 133

selects the interval between the jointed rays the primitive actino-
trichia are thereby excluded.

In such portions of the caudal fins of 6 mm. embryos,’ and in
equivalent places in later stages, are typical connective tissue
fibers mostly occurring as coarse longitudinal bundles with fine
oblique anastomoses. Single fibers occur in the intervals of the
coarser bundles. It is along these fibers and fiber bundles that
the stellate and spindle cells are disposed. These cells are readily
seen in the living fish, though the ease of observation is subject
to much variation in different individuals and to a less extent in
different portions of the same embryo.

My observations were made on living embryos immersed in sea
water, some with, some without the addition of chlorotone. In
some cases a few drops of a saturated solution of Bismark brown
were added to the sea water in which the fish was kept, the effect
of which after a time was to slightly increase the color contrast
between the connective tissue cells and surrounding structures.
The stain seemed almost inocuous, for fish could be kept in it
for several days without apparent effect on their vitality. Many
of the fish thus examined were later killed, and the fins stained
and mounted in toto, or sectioned. The various cell types seen
in life were readily recognizable in corresponding locations in the
stained preparations.

It is in life difficult or se raitertite to distinguish between the
spindle and lamellar types, though in ‘fixed’ tissue they may be
morphologically distinct. In the-living animal one can see a
stellate or a spindle cell elongate, approach and flatten itself
against a connective tissue fiber or fiber bundle, becoming some-
times so attenuated as to be scarcely distinguishable from the
fiber against which it lies; it may at any time acquire increased
thickness. Such a relation to a connective tissue fiber is shown
by the cell-b in fig. 2. The relationship is again exhibited by the
two cells shown in fig. 3, one of which a, approached a small
fiber bundle, became flattened against it, then rotated to the oppo-
site side of the fiber at 9.30 a.m., and later freed itself from the
contact. Its locomotion can be observed in relation to the chro-
matophore (ch) which served as a fairly fixed point. Similar

134 JEREMIAH S. FERGUSON

cells are frequently seen flattened against the surface of fiber
bundles, blood-vessels, or fin-rays, and exhibiting a slow stereo-
tropic locomotion. Many of these cells would seem to be identi-
eal with those which in stained preparations we are accustomed
to call lamellar cells.

That connective tissue cells exhibit a certain amount of motion
is no new observation. It has been well known since the inflam-
matory reaction to injury or infection was studied in the mesen-
tery by Arnold and others. I have observed that the extent and
rapidity of the motion varies with the cell type. The round cell,
or primitive type, presents relatively little motion, it being limited,
so far as I have observed, to the very slow projection and retrac-
tion of minute pseudopods. Even this evidence of activity seems
rather to be limited to those later phases of the cellular stage which
foreshadow the transformation of the round cells to the stellate
type of the succeeding stage. This transformation is indicated
by the fact that the motion is more noticeable near the border
of the round cell area than in its interior, and also because at the
extreme margin of such a cellular area one may by careful scru-
tiny observe an extensive alteration from round to stellate types,
some cells passing rapidly to an approximate spindle form. The
type of motion exhibited by the round cells, when observable,
is well shown by fig. 4, cells a-c being observed at the extreme
margin of the round cell area, cells d—e just within the margin,
and cells f—g well in the interior of the area.

While the general trend of cell change is from round to stellate
to spindle cells, a change may often be observed to occur in the
reverse direction, as occurred to the cell shown in fig. 5, and that
in fig. 6. Such retrograde changes are less frequently observed,
and the transformation is less extensive than are the progressive
changes from the round to the stellate forms. The retrograde
stellate phase is also more frequently of a transient character
(fig. 5). Thus, a stellate cell may by retraction of its processes
temporarily assume a spheroidal form but it soon again projects
pseudopods and regains its stellate character. Or a typical,
bipolar, spindle shaped cell may extend a third process, or even
several additional processes (figs. 2, 5 and 6), but, so far as I have

LIVING CONNECTIVE TISSUE CELLS 135

observed, such processes are limited in size and usually of short
duration. This reverse transformation may be likened to an elas-
tic rebound brought about by an inherent resistance to change of
form reacting against an impelling force which directs the trans-
formation from the round to the spindle type. The cell frequently
balks at the change, but the general trend from round to stellate
and from steilate to spindle form is inevitable.

Motion resulting in change of form is perhaps most active in the
stellate type of connective tissue cell. The general trend of this
motion seems to be indicated in fig. 5 J, in which a typical round
cell selected for observation at the margin of the round cell mass
in a pectoral fin of a 6 mm. embryo was seen within a period of
six minutes to elongate and then to pass through successive stel-
late shapes to a typical spindle form. But the succession is not
always so rapid. Stellate cells exhibit all sorts of morphological
transformations in rapid sequence (fig. 7) and this stage of con-
nective tissue development is of relatively more transient duration
than either the preceding or the succeeding stage. Moreover, the
shape of the cell is undoubtedly influenced to some extent by
its surroundings and the duration of a particular stellate, spindle
or lamellar shape may in some cases be thus determined.

Likewise, spindle cells undergo considerable transformations
in form, the most frequent of which undoubtedly result in the
lamellar shapes on the one and in the stellate on the other hand.
Because of the limitations of the microscope in the delineation of
the ‘third dimension’ it is most difficult in the colorless living
tissues to differentiate between the lamellar and spindle types of
cell but the evidence of fixed and stained tissues shows the lamel-
lar to be the more mature, the spindle the earlier type, and I
have observed nothing in the living tissues to indicate the con-
trary unless indeed it be that both types appear to be somewhat
dependent on their surroundings, for as already stated these forms,
in the same cell, seem to be more or less interchangeable. That
spindle cells frequently and freely revert to the stellate type there
is abundant evidence. There is also evidence that these cells may
be capable of still further transformations than those of mere form.
A syncytial stage in the development of connective tissue has

136 JEREMIAH S. FERGUSON

long been assumed. That this stage in its most typical form
presents those cell pictures which we are accustomed to regard
as stellate cells is well known. It is generally recognized that
this syneytial stage passes into one in which the fibers appear and
the syncytium is replaced by a tissue of cells and fibers. The
syncytial stage has been presumed to be preceded by a cellular
stage and to those who have traced the origin of the mesoderm
from the time of egg fertilization it would appear logical, even
necessary, that at a sufficiently early period a cellular character
must obtain, though Mall has questioned the preéxistence of this
cellular condition. The transformation from the cellular to the
syncytial condition has been ascribed on the basis of ‘stained sec-
tions, to either of two processes: either the syncytium arises by
incomplete division of preéxisting cells or the syncytium results
from the fusion of the preéxisting cells. That some syncytia arise
by incomplete cell division is very probably true. This appears
specially obvious in such placental tissues as the superficial cells
of the chorionic villi. I know of no convincing evidence that it
does occur in the connective tissues.

Since I have been unable to observe mitotic figures in the living
connective tissue cells of the fish which are under discussion I
cannot offer any evidence pro or con the origin of a connective
tissue syncytium by incomplete cell division. I have, however,
frequently observed a phenomenon which simulates the fusion of
processes of adjacent stellate cells after the manner of a typical
connective tissue syncytium. In figs. 2 and 8 JJ the neighboring
cells, which were at first entirely distinct and separate, were within
a brief period seen to send out processes.which on contact appar-
ently fused. But of course one cannot say without subsequent
fixation and staining of the identical cells, a process presenting the
greatest difficulties, that the fusion was actual and complete.
Even in stained sections the question is often difficult to deter-
mine. While the fusion was apparent I am not at all sure that it
was actual. Not, however, in every case when cell came into con-
tact with cell did such apparent fusion occur. This is shown in
fig. 8 I, in which processes from the cells a and b came into contact
tip to tip, yet though fusion seemed imminent it did not occur

LIVING CONNECTIVE TISSUE CELLS 137

and the contour of each cell at the point of contact remained clear
and distinct. Moreover it would seem that since connective tis-
sue cells move extensively along the surfaces of the syncytium
that syncytium could scarcely arise by fusion of its cells.

The spindle cells exhibit a certain stereotropism. They are
prone to take their position alongside a connective tissue fiber or
fiber bundle or against the surface of a blood-vessel or dermal
fin-ray. When in contact with a broad surface, such as that of a
blood-vessel or one of the lepidotrichia, the cells frequently assume
a flattened, lamellar form. This is shown by stained sections, in
which that type of cell predominates in these locations, and by the
observation of living spindle cells which frequently move up to
a blood-vessel or a fin-ray and then become so thinned out against
the surface that they finally vanish, being in the living tissue in-
distinguishable from the refraction lines which surround the larger
bodies. Again, the spindle cells very frequently move up to a
connective tissue fiber or bundle and then elongate along the nar-
row filament until, as before, the cell finally appears to vanish by
its extreme attenuation. Such a result was observed a moment
later than the recorded observation in the case of the cell shown in
he ol:

It frequently happens that the spindle cells after such elongation
again thicken to a typical spindle form, and may even throw out
other processes, but in so doing, if the cell is observed in relation
to some relatively fixed point, e.g., a joint of a dermal fin-ray, a ~~
chromatophore, or a blood-vessel, it will be seen that the cell has
changed its relative position; it has exhibited locomotion. Loco-
motion is not a distinguishing character of the spindle cell; it is
exhibited by the stellate cells, possibly also to a very limited
extent by those round cells which are only just beginning to pre-
sent pseudopod formation. But the character of the locomotion
in the several types of cells differs decidedly. In the stellate
type locomotion may take any direction and resembles a very
active amoeboid motion, processes being extended along the sur-
faces of fine fibers, then either retracted or increased in size until
the whole cell has come to occupy the place of the former process.
Though locomotion in the stellate cells is not entirely confined

138 JEREMIAH S. FERGUSON

to the direction of visible fiber lines, yet a projecting process of
such a cell often appears to envelope or to become coincident with |
a fiber. In the spindle cells locomotion is always so far as I have
observed, in the direction of the fiber lines: usually these cells
merely slide along the surface of fibers, blood-vessels and similar
structures.

I have observed that the stellate cells are more prone to lie in
relation with the finer, the spindle cells with the coarser fibers;
the coarser fibers in most cases, because of their size, being pre-
sumably fiber bundles rather than single fibers. This relation-
ship is to be expected in as much as in stained preparations one
finds the stellate cells present with those finer fibers which repre-
sent the earlier stages in fiber formation.

That fibers do lie without the cell in both embryonic and mature
connective tissue is generally conceded. That they lie within the
cell in reticular tissue, which in a way is comparable to an early
or embryonic type of connective tissue, I have recently demon-
trated by means of the Bielschowsky stain.! The types of fiber
development by fusion of intracellular granules described by
Spuler and by Lavini though perhaps not conclusively demon-
strated, at least show that certain granules which are in relation
with the first appearance of fibrils do le within the substance of
the stellate, mesodermal, connective tissue cells. Moreover, I
have found in embryonic tissues (fig. 9) just such appearances as
I have described for reticular tissue.2. By means of the Biel-
schowsky method such appearances can be shown throughout
embryonic connective tissue. I have observed them in pig
embryos, of various ages, in the limb buds, the head, the cervical
region, and in the back throughout the whole length of the embryo
from the occiput to the caudal tip, also in the umbilical cord. In
many of these locations I have made similar observations on human
embryos of older stages but in which the connective tissue was
still actively developing. One is at a loss to explain the method
by which fibers arising within the cells arrive at a location out-
side the cell body when these cells are in active motion. The

‘Am. Jour. Anat., vol. 13, page 277, 1911.
* Loe. cit., in which see especially fig. 4 and fig. 8, pages 285 and 289.

LIVING CONNECTIVE TISSUE CELLS 139

ectoplasmic theory of Hansen does not satisfactorily account for
it and its elaboration by Mall is not as specific in this particular
as one might wish. These theories do not appear to fully har-
monize with the relatively active motion and locomotion of the
connective tissue cells which I have observed in the fins of living
fish and which Harrison, Burrows and others have also to some
extent recognized in tissue cultures. The cells are not suff-
ciently quiescent to permit of endoplasmic retraction with deposit
of ectoplasmic fibers unless this retraction is rapidly performed,
in which case it should be observable in the living embryo. I
have in one or two cases suspected such a method of deposit but
have notas yet been able to convince myself that it actually occurs;
in fact I now doubt if it occurs at all.

The ectoplasmic theory presupposes that the fibers arise at the
surface of the cell. This I have found to be not always the case.
The clear delineation of fibers by the Bielschowsky method makes
it possible to follow their course within the cell more carefully
- than ever before and I find that the blackened fibrils within the
cell both in pig embryos (fig. 9)and in the fish’s fin very frequently
pass close to the nucleus, sometimes ending almost in contact with
this structure, but more frequently passing by so closely as to be
in actual contact with the nuclear membrane. I am aware that
Golowinski using the iron haematoxylin method, demonstrated
the presence of fibers at the surface of the connective tissue cells
of the umbilical cord and that the apparent relation to the nu-
cleus was explained by him as due to obliquity of section. But I
have not in my preparations been able to convince myself of the
adequacy of this explanation. I have found fibers to be not
always at the surface of the cell, they may and frequently do pene-
trate entirely through the cytoplasm of the cell, as I have previ-
ously described? for mature reticular tissue. In the developing
connective tissue, as well as in reticular tissue, such penetration of
cells by the fibers is so frequent as to appear quite characteristic.
It seems to me that the intimate relation of connective tissue cells
and fibers in embryonic tissues can only be accounted for by tak-

3 Loe. cit., see fig. 10, page 293.

140 JEREMIAH S. FERGUSON

ing cognizance of the plasticity of the connective tissue cellular
cytoplasm, and also of the active motion of connective tissue cells
during the period in which the fibers are being formed, so that the
finer connective tissue fibers become, by the cellular activity,
embedded in the plastic cytoplasm of the cells during their stereo-
tropic locomotion. The plastic character of the cellular cyto-
plasm is admirably shown by the rapid changes in form of the
connective tissue cells in the fins of living fish embryos.

I have already stated that the spindle cells of connective tissue
in the fins of living fish undergo active locomotion. In fact this
seems to be a most prominent function of the spindle cell type.
Most frequently the cell glides along connective tissue fibers which
often appear to be thus partially enveloped by the cytoplasm.
In recording this-stereotropism I am able to corroborate, for the
living cells of embryo fish, the observations of Harrison on tissue
cultures in which he finds that the connective tissue cells are spe-
cially prone to follow along the surface of fixed objects. Such ob-
jects in normal living subjects are most frequently the connective -
tissue fibers and fiber-bundles already deposited, though as pre-
viously stated, I have also observed connective tissue cells moving
along the surface of the dermal fin rays and of blood-vessels.
In this form of activity the cells adapt themselves more or less to
the shape of the surface along which they are moving. They
wrap themselves about or rotate around the finer fibers (fig. 3)
and they flatten themselves against the larger objects (figs. 2,
3and5). In this attenuated condition they still move along the
surface of fibers, often at a considerable rate of speed. One such
cell I have recorded in a preliminary communication‘ was found in
ten minutes to have covered a distance of 50y, a rate of 1p» in
every twelve seconds.

The striking similarity of the living, spindle, connective tissue
cells to those of fixed tissue is indicated in fig. 10 which shows
several such cells from a 100 mm. pig embryo. The magnifica-
tion is the same as that used for the observation of the living cells.
The similarity in the form of the cell and the relation of cells to
fibers is apparent on comparison with the preceding figures. One

‘ Biol. Bull., vol. 21, page 272, fig. 2, 1931.

LIVING CONNECTIVE TISSUE CELLS 141

ean scarcely avoid the interpretation that the cells shown in fig.
10 were at the moment of ‘fixation’ moving along the surface of

_ the fiber bundles against which they lie.

The pronounced morphological relation between the connective
tissue cells and fibers cannot but have an equally close functional
relation. What those functional relations may be we are not
now possessed of the data to fully determine.

Further studies will be necessary to fully understand the part
played by the active moving connective tissue cells in the pro-
duction and growth of the collaginous fibers. So far as they are
now determined the essential phases of the process in which the
connective tissue cells are concerned appear to be: (1) the connec-
tive tissue arises from a primitive mass of round mesenchymal
cells; (2) there is a change of form from round to stellate, to
spindle, and eventually to lamellar cells; (3) certain fibers seem
first to appear within the cells, possibly at their surface (Hansen,
Golowinski); (4) there is formed a reticulum pervading the inter-
cellular ground substance whose fibers may be, though they not
necessarily are, identical with those first arising within the ‘cell;
(5) coincident with the origin of fibers there begin amoeboid move-
ments in the stellate and spindle cells; (6) there is an increase in
size and number of fibers in the reticulum and they aggregate
into bundles, synchronously with which first the stellate and later
the spindle cells move along the surface of the fiber and fiber
bundles.

In conclusion I desire to express my sincere thanks to the
Marine Biological Laboratory at Woods Hole, Massachusetts,
for the opportunities so kindly placed at my disposal.

BIBLIOGRAPHY

Arnotp 1893 Arch. f. path. Anat., vol. 132, p. 502.

Bout, Franz 1872 Arch. f. mikr. Anat., vol. 8, p. 28

Burrows, M.T, 1911 Jour. Exp. Zool., vol. 10, p. 63.

CARREL, ALEXIS, AND Burrows, M.T. 1910 Jour. Am. Med. Assoc., vol. 55, 1379.
1911 Jour. Expr. Med., vol. 13, p. 416.

Ferauson, J.S. 1911 Am. Jour. Anat., vol. 12, p. 277.
1911 Biol. Bull. vol. 21, p. 272.

FLEMMING 1891 Festschr. f. R. Virchow.
1897 Arch. f. Anat., p. 171.
1906 O. Hertwig’s Handbuch. d. vergleich. u. exper. Entwickl. d.
Wirbeltiere, vol. 2, p. 1.
GoLowinsk1 1907 Anat. Hefte, vol. 33, p. 205.
Goopricu E. 8. 1904 Quart. Jour. Mic. Sc., vol. 47, p. 465.
Hansen, F.C. C. 1899 Anat. Anz., vol. 16, p. 417.
Harrison, R.G. 1893 Arch. f. mik. Anat., vol. 43.
1895 Anat. Anz., vol. 10, p. 138.
1910 Jour. Exp. Zool., vol. 9, p. 787.
1911 Science, vol. 34, p. 279.
HENLE, J. 1841 Allgemeine Anat., S. 197, u. 397.
v. Korrr 1907 Ergebnisse d. Anat. u. Entwickel., vol. 17, p. 247.
Lewis, Marcaret R. anp W.H. 1911 Anat. Record, vol. 5, p. 377.
Lrvint, F. 1909 Monitore zool. Ital., vol. 20, p. 225.
Matt, F. P. 1902 Amer. Jour. Anat., vol. 1, p. 329.
MERKEL, Fr. 1895° Verhandl. d. Anat. Gesellsch., vol. 9, p. 41.
Scuwann, THEODOR 1839 Morphological Researches, trnsl., 1847.
SpuLter 1897 Anat. Hefte, vol.7, p. 115.
VALENTIN R. Wagner’s Handworterbuch der. Physiologie, vol. 1, p. 670.
Vircuow, Rupotr 1852 Verhandl. d. Phys. Med. Ges., vol. 2, pp. 150, 314.
1860 Cellular Pathology, transl. from 2d German ed., p. 43.
ZieGLER 1903 General Pathology, transl. from 10th German ed., p. 353.

PLATE 1

EXPLANATION OF FIGURES

1 From aFundulus embryo, of 5.5 mm. total length, showing beginning amoe-
boid movements of two cells, a and b, on the border of the round cell area at
the posterior extremity of the ventral median fin. The observation extends over
a period of fifteen minutes. The last seven drawings were made without change of
focus for the purpose of eliminating variation in form due to the examination of
different levels. a, b, two connective tissue cells. Ch, prominence on the sur-
face of a chromatophore, the body of the cell is not represented. The numerals
in this and succeeding figures indicate the exact time at which each recorded
drawing was completed.

2 Connective tissue cells, one of which, a, exhibits transformation from a
spindle to a stellate type, and another, b, becomes flattened against a connective
tissue fiber. There was an apparent anastomosis between cell a and the proto-
plasmic process p of an adjacent cell. f, connective tissue fiber; 7, margin of a
joint of a fin ray, giving a fixed point in relation to which locomotion may be ca
mined. Other letters and numerals as in the preceding figures.

3 ‘Two connective tissue cells exhibiting some locomotion. One of these, a,
assumed a lamellar like relation to a fiber bundle while rotating about it. At
9.25 A.M. this cell became momentarily so thin as to almost escape observation.
Ch, Ch’, chromatophores. Other letters and numerals as in the preceding figures.

142

LIVING CONNECTIVE TISSUE CELLS PLATE 1
JEREMIAH 8. FERGUSON

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423 425 426 427 429

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THE AMERICAN JOURNAL OF ANATOMY, VOL, 13, No. 2

143

PLATE 2
EXPLANATION OF FIGURES

4 Amoeboid motien resulting in change of form exhibited by connective tissue
cells of the primitive or ‘round’ type. J, cells a-c, from the extreme margin; JJ,
cells d-e, from just within the margin; and JJJ, cells f-g, from the interior of a
round cell area.. J and JJ from the pectoral, J7J from the caudal fin. Numerals
as in the preceding figures.

5 I, transformation of a round to a spindle cell in the pectoral fin of a Fun-
dulus embryo 6 mm. long, 20 days after fertilization, 11 days after hatching.
From 4.20 to 4.22 p.m. there was in this cell an apparently retrograde change from
spindle to stellate form but at 4.24 p.m. this had been proven temporary. JJ,
transition of a stellate cell to a temporary spindleform. Letters and numerals as
in preceding figures

6 Apparent retrograde change from spindle to a stellate form in a cell under-
going rather slow locomotion. The stellate phase of such cells is nearly always
temporary. From the same embryo as fig. 5; cap., blood-capillary. Other letters
and numerals as in the preceding figures.

144

PLATE 2

LIVING CONNECTIVE TISSUE CELLS
JEREMIAH 8, FERGUSON

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PLATE 3

EXPLANATION OF FIGURE

7 Stellate connective tissue cells from the fins of four embryo fish, J-IV.
exhibiting rapid change of form. Owing to difficulties of observation it is not
possible to make drawings oftener than at 1 to 3 minute intervals; hence, the
actual changes of form were much more frequent than the record shows. A-K.
nine connective tissue cells. Numerals as in the preceding figures.

146

LIVING CONNECTIVE TISSUE CELLS PLATE 3
JEREMIAH S. FERGUSON

PLATE 4

EXPLANATION OF FIGURES

8 Stellate connective tissue cells exhibiting locomotion and, on contact, appar-
ent fusion. J, from the pectoral fin of a 10 mm. Fundulusembryo. The fusion
between cellsaand 6 is apparent only. bandc appear to form part of the anas-
tomosing syncytium. JJ,from the caudal fin of a6 mm. Fundulusembryo. Cells
e and g on coming into contact at 4.44 p.m. apparently fused after the manner of the
cells which form the delicate early connective tissue syncytium. Letters and
numerals as in the preceding figures.

9 Stellate connective tissue cells in the subectodermal mesenchymal syncy-
tium of a 25 mm. embryo pig. Fibrils pass through the cells very close to the
nucleus. Bielschowsky stain.

10 Connective tissue cells from the praevertebral (J) and intermuscular (7)
connective tissue of a 100 mm. pig embryo. The form of the cells and their
contact relations to adjacent connective tissue and muscle fibers is strikingly simi-
lar to the amoeboid connective tissue cells of the living fish embryo. The appear-
ance suggests that such cells were quite probably moving along the surfaces with
which they are in contact.

148

LIVING CONNECTIVE TISSUE CELLS PLATE 4
JEREMIAH 8S. FERGUSON

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The Relation of Muscle Cell to Muscle Fibre in Voluntary
striped Muscle.

By W. M. Baldwin,
Cornell University Medical College New York City.

From the Biological Laboratory at Bonn.
of
With table VII and VIII.
(Der Redaktion zugegangen am 19. Juli 1912.)

It may be stated in general terms that our present conception
of a voluntary muscle fibre is that of an enormously large, multi-
nucleated cell invested by a cell membrane or sarcolemma and con-
taining the several elements of contractility. Or, as a recent text-
book of histology phrases it, ,,Die quergestreifte Muskelfaser wire
also als ein vielkerniges, fadenformiges Plasmodium mit deutlicher
Zellmembran zu bezeichnen“ (Sonorra). That this “cell membrane”
invests, in addition to the muscle fibrillae and muscle nuclei, at
least two entirely different forms of protoplasm,') so far as their
morphological appearances are concerned, there has been but little
question but at the same time comparatively little mention in the
literature of this feature. The one form immediately invests the
nuclei and possesses the characteristics of cellular protoplasm as
elsewhere observed; a granule-laden spongioplasm network with
interstices of clear hyaloplasm. These general features are wanting
in the other form, i. e, the sarcoplasm of the muscle fibre.

') Many of the published plates of such investigators as HEIDEN-
HAIN, HoLM REN, NusspauM. SCHNEIDEK, MARCEAU, WERNER, PRE-
NANT, etc. represent these differences most clearly.

The Relation of Muscle Cell to Muscle Fibre in Voluntary etc. 131

These remarks, naturally, apply more particularly to the adult
fibre. In the developing fibres the quantity of protoplasm investing
the nuclei is relatively greater and much more coarsely granular.
During this period it possesses apparently very intimate develop-
mental relations to the muscle fibrillae and to the as since
its quantity diminishes as the latter increases.

Investigators who have worked upon the structure of striped
muscle have in general regarded a close functional relationship as
existing between the sarcoplasm and muscle fibrillae on the one
hand and between the muscle nuclei and adjacent differentiated
protoplasm on the other, yet the exact morphological connection
existing between the latter and the sarcoplasm has been but little
examined beyond the general assumption that the two stood in
intimate structural contiguity with each other.

It was with the express intention of carefully considering this
relationship that I undertook the study of the muscle fibre, con-
fining my efforts for the present to the voluntary cross-striated
muscles of the tadpole, frog, chicken, calf, white mouse, gray mouse,
and cat. I used for the investigation specimens of the extrinsic
muscles of the eyeball, intercostales, rectus abdominalis, latissimus
dorsi, adductors of the thigh, sartorius, flexors of the foot together
with the caudal muscles of the tadpole. These various muscles
were fixed in sublimate, FLemmine’s solution, Mreves’s solution, and
in 96°/, alcohol. They were imbedded in paraffin, some after
Scuutrze’s collodion and cedar oil method, and sectioned longi-
tudinally, obliquely, and transversely in thicknesses varying from
2 to 5 mu. As stains I made use of picric acid alcohol, fuchsin S,
picro-fuchsin, GaGe’s chloral - hematoxylin, Scuuurzer’s alcoholic-
hematoxylin, eosin-xylene, and an alkaline ferric-tannate solution.

The sketches which I present, while they were made from
actual specimens, are accurate, however, only so far as concerns
the exact relationship of the protoplasm immediately investing the
muscle nuclei to the muscle fibrillae and to the sarcoplasm of the muscle
fibre. That is to say, inasmuch as the structural features of these
other muscle elements did not bear directly upon the subject of
the investigation, I made no effort, beyond a mere approximation
of accuracy, to delineate either their structure and their relationship
to each other or the ultimate structural features of the muscle
nuclei. Accordingly, these figures are only to be regarded as
authoritative with these two qualifications.

3

132 W..M. Baupwiy,

In most instances, I have represented only a single muscle
nucleus with the adjacent muscle fibrillae and sarcoplasm. The
relations shown were found to be characteristically present throughout
the entire series. For the demonstration of the facts, however, I
deemed it sufficient to represent only a single nucleus and the
immediately adjacent muscle fibrillae. Hence these are not to be
regarded as exceptional instances.

I have represented in Fig. I a portion of a fibre of an extrinsic
muscle of the eyeball of a white mouse fourteen days old. At this
age the eyelids are open. The fibre is invested by its sarcolemma
lying upon which at one end there are several connective tissue
cells and fibres. In the middle of the muscle fibre a nucleus
surrounded by a considerable amount of protoplasm is shown. The
characteristic features of cellular protoplasm as generally recognized
are to be observed in connection with this protoplasm, i. e., strands
of granule-laden spongioplasm composing a network which encloses
clear hyaloplasm. The exact extent of this protoplasm in either
direction is not shown, because of its passage out of the level of
the oblique section. It is to be observed, however, that the struc-
ture of this protoplasm presents a marked contrast to that of the
relatively uniformly and faintly staining sarcoplasm of the muscle
fibre. It is further to be noted that the latter is sharply delimited
from the spongioplasm network by the presence of a distinct mem-
brane. The fibrils of spongioplasm can be observed to be attached
to the internal surface of this membrane. That the existence of
this membrane, as a distinct structural entity limiting the proto-
plasm, is not merely an appearance owing to the presence of over-
lying contingent muscle fibrillae is most clearly demonstrated by
reason of the obliquity of the section. The relatively clear spaces
intervening between the obliquely-sectioned fibrillae are observed
to be bridged over by this structure. This particular feature is
more clearly represented in the lower end of the section. In brief,
then, we are dealing here with features which represent every
characteristic of cell structure, a nucleus, cell protoplasm, with
spongioplasm and hyaloplasm, and a cell membrane, all imbedded
in a voluntary muscle fibre.

The clear spaces observed within the cell body immediately
above and below the level of the nucleus I have regarded as arte-
factitious, referable as much to a retraction of the nucleus as to a
corresponding shrinkage of the cellular protoplasm. At the same
time attention must be called to the fact. that in, spite of these

The Relation of Muscle Cell to Muscle Fibre in Voluntary etc. 133

circumstances there is no indication in the section whatever either
of a retraction of the spongioplasm from the cell wall or of the
cell wall from the sarcoplasm outlying it. In many of the specimens,
I have identified spongioplasm fibrillae which were attached to the
nuclear membrane by one end and by the other to the cell wall.

I desire particularly to call attention to the relation ‘of the
cell wall to the nuclear membrane at the level of the nucleus. The
two lie so close together as to be apparently fused. It is owing
to the juxtaposition of these two structures that, when a muscle
fibre presenting such a cell is studied in cross-section at the level —
of the nucleus, the cell membrane escapes observation altogether
and the nucleus appears to lie imbedded in and in direct contact
with the sarcoplasm. My own transverse sections of this same
muscle and of all of the other muscles which I have studied, furnish
ample evidence of this appearence, yet in those transverse sections
in which, either as the result of the agents used in the preparation
of the specimen or where, as the result of a tear in the muscle
fibre, the nucleus was pulled away from the sarcoplasm, the pre-
sence of the cell membrane was then manifested and its existence
at the level of the nucleus established. Furthermore, when tran-
sections which exhibit no traces whatever of shrinkage are studied
either directly above or immediately below the level of the nucleus,
the cell membrane can be seen most clearly and the fact observed,
as well that it passes completely and uninterruptedly around the
cell protoplasm.

In fig. II I have represented an oblique section of a similar
muscle fibre presenting appearances comparable to those just
described. A muscle cell occupies the middle of the fibre. Several
connective tissue cells and fibrillae lie upon the sarcolemma. Very
often in studying fibres of the extrinsic eye muscles of the white
mouse, I have encountered specimens in which, apart from .the
characteristic muscle spindles, several muscle nuclei were closely
associated with each other in the middle of the fibre. I have
found as many as five nuclei so situated, imbedded in a protoplasmic
mass which extended in the long axis of the fibre. This mass was
everywhere marked off from the sarcoplasm by a membrane, similar
in morphological structure and in relations to that which I have
described above in connection with fig. I.

The question naturally arising in such instances was whether
such .a protoplasmic mass represented a single muscle cell containing
several nuclei or whether the nuclei each represented distinct cells

134 W. M. BaLpwin,

whose boundaries, because of their thinness, were not to be separately
differentiated. In most instances I was not able to answer this
question even in specimens studied under the best conditions of
fixation and of stain. The specimen at hand furnishes, however, a
suggestion of the probable solution of the difficulty. I can commit
myself, however, to a positive statement regarding the general
problem only so far as this one cell in particular is concerned.
The protoplasm occupying the upper portion of the sketch and
investing the nucleus there situated, is marked off from that found
in the lower end by an obliquely-placed membrane, which, though
faintly stained and somewhat indistinct, has the morphological
appearance of a true cell wall. The lower mass of protoplasm
contains as the next section in the series shows, also a nucleus.

The presence of such elongated masses of protoplasm is not
restricted to the mouse. They can be observed as well in the
extrinsic eye muscles of the three-weeks chicken. As yet, however,
I have not detected these masses in the adult muscles of the thigh,
leg, abdomen, or thorax of the white mouse, frog, chicken, gray
mouse, or cat. In such muscles the nuclei are usually isolated from
each other, while the quantity of protoplasm immediately investing
them is relatively much less in amount.

Figure ILI represents a portion of a thigh muscle of an adult
frog, showing features similar to those observed in connection with
the white mouse muscles. The section was cut obliquely; conse-
quently the muscle fibrillae cannot be traced for any considerable
distance. I have represented in the sketch a single muscle cell
with the adjacent fibrillae. This cell was imbedded in the muscle
fibre and bore no immediate structural relationship to the sarco-
lemma. By reason of the obliquity of the section the internal
surface of the cell membrane can be seen at the upper end of the
cell. There is no question as to its presence or as to its continuity.
As a distinct limiting membrane it can be traced from the vicinity
of the fibrillae situated upon one side of the cell to those on the
other,

Very often it is difficult to differentiate between the telo-
phragma (Z) lines of the muscle fibrillae and the reflected cell
membrane at the poles of the cell. In this particular specimen,
however, this difficulty is lessened by reason of the fact that the
features of cross-striation of the muscle are not distinctly marked.
The cell wall, accordingly, can be seen all the more clearly. By
reason also of the alcoholic hematoxylin stain used on this prepa-

The Relation of Muscle Cell to Muscle Fibre in Voluntary etc. 135

ration the fibrillae are much lighter in color than the cell wall.
Hence the possibility in a longitudinal section of mistaking the
border of a muscle fibril for the cut edge of the cell wall is also
eliminated. The cell membrane is readily demonstrable as a formed
structure separated from the muscle fibrillae by a narrow interval
of sarcoplasm. What is, however, a much more significant fact
pointing towards the presence of the cell wall is, as the sketch
shows, the presence of several strands of spongioplasm which
terminate upon this cell wall. In no cell which I have studied
have I ever seen a fibril of spongioplasm terminating upon a muscle
fibril.

At the lower end of the cell two, sectioned, muscle fibrillae lie
upon the external surface of the cell wall. These fibrillae passed
out of the plane of the section in their passage around the cell
body. Several published plates would lead us to infer that in some
instances at least, muscle fibrillae terminated at a muscle cell. I
have never observed such an instance, however, in my own
specimens. In every section the fibrillae diverged from each other
in order to pass uninterruptedly beyond the cell.

The remarks, which I made regarding the relation of the cell
wall to the nuclear membrane opposite the center of the nucleus
in connection with the mouse muscles, are to be emphasized at this
place with regard to the frog muscle. The cell membrane every-
where separates the nuclear membrane from the sarcoplasm. And,
likewise, in those specimens, where a shrinkage of the nucleus has
taken place, its presence is most readily made out.

In some of the transverse sections of frog muscle the muscle
fibrillae were torn away from each other. I have represented such
a section in fig. IV. The sketch was taken at an optical level a
little above the center of the nucleus. This shows a cell located
in the middle of a muscle fibre. The muscle fibrillae lying above
and to the left were undoubtedly torn away from a more intimate
relation with the cell. From a careful study of the preparation,
I concluded that the nucleus as well had been removed slightly in
a direction upwards and to the left. In this direction the cell wall
itself was torn. It remained intact, however, and in its proper
position relative to the muscle fibrillae and sarcoplasm upon the
other two sides. At the left side of the cell the edge of the cell
wall can be seen to project from its attached position to the sarco-
plasm into the clear space occasioned by the tear in the muscle
fibre. It is further to be observed that the part projecting into

136 W. M. Banpwin,

this tear is unaccompanied by sarcoplasm. These features are less
clearly outlined upon the right side of the cell. The specimen
furnishes to my mind the most conclusive evidence as to the
existence of the cell membrane as a distinct, independent, morpho-
logical structure.

A cell membrane is characteristically associated not only with
those muscle nuclei which are located in the middle of a muscle
fibre, but as well with peripheral-lying nuclei. I have observed
the same relationship of nucleus to protoplasm, of protoplasm to
cell wall, and of cell wall to sarcoplasm in connection with those
nuclei which are applied to the sarcolemma. The cell membrane
everywhere separates the sarcoplasm from the spongioplasm network
of the cell. Upon that surface of the cell which is directly applied
to the perimysium, however, the cell wall fuses with that structure
and loses its identity.

In figs. V and VI are represented portions of muscle fibres
removed from the thigh of an adult cat. Both of these cells lay
upon the periphery of the muscle fibre in contact with the peri-
mysium. The fibre in fig. V was sectioned longitudinally and that
in fig. VI obliquely. A fold occurring at the level of the nucleus
accounts for the transected fibrillae in the first. In both instances
the cell membrane can be seen distinctly. It separates the cellular
protoplasm containing the nucleus from the contractile elements of
the muscle fibre. Upon that side of the cell which is applied to
the perimysium it is fused with this structure and cannot be traced.

In the study of similar preparations presenting these features
it was necessary to differentiate muscle nuclei applied directly to
the internal surface of the perimysium from those connective tissue
cells lying upon the external surface of that. structure and from
those which were, to all appearances, in the perimysium itself. *)
The fact is quite self-evident in the figures, particularly in fig. VI,
that the nucleus is imbedded among the muscle fibrillae. That is
to say, this relation is more intimate than would be expected were
the cell situated outside of the sarcolemma but causing an indentation

‘) This last statement requires some qualifications in view of the
recent work of SCHIEFFERDECKER upon the sarcolemma. The nuclei to
which | refer are so situated within what appears to be the sarcolemma
that this latter structure splits apparently to enclose them. .At any rate
it is not possible to see any formed structure between such nuclei and
the relatively clear sarcoplasm. I will comment more at length upon
these special features, however, in a forth-coming paper.

The Relation of Muscle Cell to Muscle Fibre in Voluntary etc. 137

of the muscle fibre. Furthermore, the morphological appearances
of these cell walls are precisely similar to those noted before as
characteristic of cells found imbedded among the fibrillae in the
middle of a muscle fibre. Again, a difference can very readily be
observed between these peripheral muscle cells and what, to all
appearances, are cells located in the perimysium. The latter are
small and spindle-shaped, with a compact and deeply-staining
nucleus surrounded by a relatively small amount of dense and uni-
formly deeply-stained protoplasm.

From the study of extrinsic muscles of the eyeball of a calf,
I was convinced of the presence of a cell membrane similar in
relations and in its morphology to that which I had observed in
the other vertebrates.

In fig. VII I have represented an entire muscle fibre cut
transversely. The sketch was taken a little above the level of the
center of the nucleus. At the latter level the cell membrane was
not visible as a separate structure owing, as I have previously
mentioned, to its contact with the nuclear outline. At the level
represented, however, it is readily seen. I desire to direct parti-
cular attention to its relation to the muscle fibrillae. I have never
found it in direct contact with these structures. An interval of
sarcoplasm always intervenes between the two. In general terms
it may be stated that this intervening layer of sarcoplasm averages
in thickness to the cross diameter of a muscle fibril.

The sections of latissimus dorsi of the three-week-old chicken
also show the same general features of cell protoplasm and cell
wall. In fig. VIII I have represented a transection of an entire
muscle fibre under the same conditions as those in fig. VII. The
cell wall lies at a little distance from the adjacent fibrillae. It
stains very deeply in contrast to the intervening sarcoplasm. At
levels above and below that of the nucleus fibrils of spongioplasm
can be traced to its internal surface where they find an attachment.

In the study of the longitudinal preparations of striped muscle
fibres, there are introduced several factors which render the identi-
fication of the wall of the muscle cells particularly difficult. First
among these to be mentioned, is the presence of the parallel-running
muscle fibrillae. The free edge of a fibril may be very easily
mistaken for the cell membrane. What is more significant, however,
is the overlying or underlying of the membrane by these fibrillae
thereby overshadowing the delicate cetl outline. Still another diffi-
culty is encountered at the extremities of the cells where the telo-

138 W. M. Baupwiy,

phragma lines bridge over the narrowed cell protoplasm. Dependent
upon this latter factor is the difficulty in the determination of the
cell extent particularly enhanced. Then, lastly, scattered among
the muscle fibres many connective tissue cells are found whose long
axis corresponds very often to that of the muscle cells and, accor-
dingly, to that of the muscle fibre. In their general outline, in the
morphological characteristics of their protoplasm, and in that of
their nucleus, as well, these cells often resemble the muscle cells
very closely. Therefore, in the instance of such cells occurring
upon those aspects of the muscle fibre either facing towards, or
turned away from the observer it is, at times, not easy to diffe-
rentiate the cells as muscle cells or as connective tissue cells. In
other words to say whether the cells lie inside of the sarcolemma
or outside of it.

In fig. IX I have represented a portion of a fibre from an
extrinsic muscle of the eyeball of a calf. Two muscle cells, whose
nuclei indent -the longitudinally-cut fibre, are to be seen. The
marked morphological differences between the protoplasm of these
cells and the sarcoplasm of the fibre are most apparent. Notwith-
standing, this fact by reason of the presence of the parallel muscle
fibrillae, it is exceedingly difficult in this specimen to determine
the exact limits of this protoplasm. In specimens stained with
alcoholic hematoxylin and then properly extracted, however, the
differentiation of cell membrane from fibrillae is rendered possible.
Serving as good controls for such longitudinal sections are obliquely-
sectioned preparations, for in such the cell wall can most readily
be distinguished apart from the fibrillae.

In focusing down through this particular specimen, I encountered,
first, the telophragma lines with the muscle fibrillae upon the upper
aspect of the fibre, then the cell nucleus and protoplasm, and lastly,
the same lines upon the under aspect of the fibre. Hence I was
most positive that these were muscle cells within the fibre and not
connective tissue cells either overlying or underlying the muscle
fibre border.')

At this place again, I desire to point out the closeness of
approximation of the nuclei to the muscle fibrillae. The latter are

") Ihave projected the telophragma lines upon the same plane with
the nucleus and cell protoplasm. They do not encircle the peripheral
cells, however, nor do they traverse the cell protoplasm. J have made
these points the subject of a forth-coming paper on the sarcolemma.

The Relation of Muscle Cell to Muscle Fibre in Voluntary ete. 139

diverted out of their course slightly at its level. At this same
level the cell wall and nuclear membrane are in contact with each
other and are not separately distinguishable.

The structural relationships noted in connection with the
calf’s-eye muscle, I observed as well in the latissimus dorsi muscle
of the three weeks chicken. Fig. X represents a portion of a fibre
from this muscle. This is an alcoholic hematoxylin preparation
which, notwithstanding the proximity of the muscle fibrillae, demon-
strates the cell outlines very clearly. That the two cells seen
were muscle cells and not connective-tissue cells located without
the perimysium, I established by determining their position a
to the telophragma lines.

A muscle fibre from an extrinsic muscle of the eyeball of a
white mouse is represented in fig. XI. This is a chloral-hematoxylin
preparation. The outlines of the two cells upon the right of the
figure are not clearly seen. Both of these cells, however, are
muscle cells. In the instance of the single cell situated upon the
left border of the fibre, we can note a morphological similarity to
those represented in fig. IX.

In the specimens of leg, intercostal, and rectus abdominalis
muscles of the adult mouse, the muscle cells are relatively less
numerous, yet the cell membrane is everywhere clearly defined.

I present fig. XII for the purpose of demontrating one of the
difficulties encountered in establishing the extent of the protoplasm
of a cell in a longitudinal direction. The specimen is a tail muscle
of a tadpole about 5,0 cm long. In either direction, as is shown,
the cell protoplasm is drawn out to a pointed extremity lying
between parallel-running muscle fibrillae, and occupying a space
about equal in cross-diameter to that of the average interval existing
between adjacent fibrillae. It is readily understood from a careful
study of the figure how the elements of cross-striation, where over-
lying the narrow. pointed extremity of the cell, are very readily con-
fused with that extremity. Indeed, for this reason, it is extremely
difficult to determine in any given instance the true limit of the cell.

In conclusion it may be stated in general terms that the cell
membrane is seen to best advantage in those oblique sections which
are cut ata thickness of 2 w and are stained with either alcoholic
hematoxylin or with alkaline ferric tannate, and in such instances
where the cut edge of the membrane, owing to the obliquity of the
receding cell wall, is underlaid by nothing but the clear hyaloplasm
of the cell. In many such instances where the staining is of the

140 W. M. Batpwiy,

proper depth the internal surface of the membrane itself can be
made out and its continuity, delicacy, and similarity to other cell
walls observed. With the hematoxylin stain, the clear fibrils of
spongioplasm can be seen to be attached to its internal surface.
The interval of sarcoplasm existing between the muscle fibrillae
and the cell membrane can also be noted. That the membrane is
more than the thickened edge of the sarcoplasm seems to my mind
to be established by its staining reaction, its presence as a distinct
lamina separate from the sarcoplasm, in those instances where the
muscle fibre has been torn, and by the attachment of fibrils of
spongioplasm to its internal surface. That it is not an artefact
produced by the shrinkage of protoplasm toward and upon the edge
of the sarcoplasm, I believe, because it can be clearly observed in
specimens where there is no indication whatever of shrinkage else-
where in the tissues. :

Concerning the query as to whether all of the nuclei with their
surrounding protoplasm are invested with a cell wall, I-can answer,
that in all of the specimens which I have as yet studied and which
were properly cut and stained, I have been able to make out the
cell wall. Because of this fact, in such muscles as the intercostales
which | have studied in their entirety, I consider that the muscle
fibrillae are to be regarded as extracellular and not, as we have
heretofore believed, intracellular. In the instance of the other
voluntary striped muscles, since I have as yet observed no cell
which did not possess a definite cell wall, I feel reasonably sure,
though I have not as yet sectioned and studied an adult muscle as
the sartorius or latissimus dorsi of the cat in its entirety, that the
generalization, that the voluntary, striped-muscle fibrillae of the
adult should be regarded as extracellular, is justifiable.

The apparent reason why this membrane has been overlooked
by the very great number of investigators working upon muscle is
referable not so much, I believe, to a fixation method fault as to a
staining defect. I have, for instance, restained slides which were
at first stained with eosin, fuchsin S, picro-fuchsin, and even with
alcoholic hematoxylin, in which, however, the extraction of the
stain from the muscle fibrillae was relatively not sufficient, and in
which, accordingly, the cell membrane could not be seen, and in
the same section after treatment with alcoholic hematoxylin and
after a proper extraction of that stain, I have been able to define
the membrane most clearly.

In order to guard against the false’ appearances produced by

The Relation of Muscle Cell to Muscle Fibre in Voluntary etc. 141

shrinkage in stained sections, I studied many preparations of
living muscle of the frog and the tadpole. In all of them the muscle
cell outlines were very clearly seen. In fig. XIII I have represented
a single muscle cell with the adjacent fibrillae from a thigh muscle
of an adult frog. The outline of this sketch was made within ten
minutes from the time that the portion of muscle was removed from
the thigh of the living animal. No fixatives or other chemicals
producing shinkage were employed on the specimen. The presump-
tion is fair, I believe, that the sketch represents the condition as
seen in a living muscle fibre. The outline of the cell body with
its distinct cell wall is most clearly seen. The muscle fibrillae are
very lightly stained with dilute methylene blue in comparison to
the protoplasm of the cell body. The fibrils of spongioplasm can
be observed to be attached to the cell wall. This preparation
eliminates all doubt as to the presence of a definite cell wall in
the living muscle and which is not produced in fixed and stained
sections by artefactitious changes. *)

Many workers, such as Maui, FuemMinG, SpaLnTEHOLZ, HANSEN,
and others, have observed that the anlages of connective tissue
fibrils are located within the genetic cell bodies. An adult fibril
of such tissues is, however, either wholly extracellular or partly
extracellular and partly intracellular. The names of Rouuert, Tour-
NEAUX, Meves and Fercuson, in addition, may be mentioned in support
of this latter statement.?) The same developmental sequence is
observed in the instance of elastic tissue fibrillae. The work of
Harrison may also be cited, — ,,Die wichtigste Tatsache aus den
vorgegangenen Beobachtungen ist, dab diese Skeletteile — die
Hornfiden und Flossenstrahlen (of teleosts) — keinen intercelluliren
Ursprung haben, sondern das Ergebnis einer direkten Umbildung
yon gewissen Zellenteilen darstellen, welche yon dem Mesenchym
abzuleiten sind.“ The anlages of muscle fibrillae, as well, have an

1) In reviewing the literature it is not surprising to find that many
of the published plates of striped muscle structure present what appears
to be a muscle cell membrane. HEIDENHAIN’s .,Plasma und Zelle“, con-
tains such figures, as do also the following references to invertebrate
muscle, Lrypia’s ,Untersuchungen zur Anatomie und Histologie der
Tiere“, of 1883 and NussBpaum’s ,Anatomische Studien an californischen
Cirripedien“ of 1890. It is a remarkable fact, however, that none of
these authors recognized the significance of the membrane nor the extra-
cellular character of the muscle fibrillae.

*) SPALTEHOLZ has recently questioned this view, holding that the
fibrillae of the adult tissues are still intracellular.

142 W. M. Batpwiy,

intracellular position as HerpEnnaln, GODLEWSKI, Mreves, Marceau,
BARDEEN, among others have demonstrated. My own studies of the
adult, voluntary, striated, muscle fibres show that the voluntary
muscle fibrillae have an extracellular position. The intermediate
steps in histo-myo-genesis are as yet incomplete, still with the facts
at hand derived from these two extremes, a seeming parallelism can
be drawn between the histogenesis of the connective and elastic
tissues:on the one hand and that of the voluntary striped muscle
fibrillae on the other. Accordingly. it would appear to be justifiable
to attribute to this form of muscle fibre a position among the
connective tissue group of structures and not to regard it as a
purely cellular structure. My conception of a voluntary striped
muscle fibre is, therefore, not that of a gigantic, multinucleated cell,
but rather of a composite contractile structure enveloped by a
sheath, the sarcolemma, which encloses, in addition to the elements
of contractility, muscle fibrillae, sarcoplasm, etc. muscle cells,
which cells present all of the recognized features of general cell
structure, cell membrane. cell protoplasm, consisting of spongioplasm
and hyaloplasm, and a nucleus containing nucleoli. +)

Zusammenfassung.

Die quergestreifte Muskelsubstanz ist nur anfangs eine intra-
cellulare Bildung und wird spiter extracellular verlagert, so dab
Muskelzellen und fibrillire Substanz mit dem zugehérigen Sarko-
plasma getrennte Bestandteile werden. Die Histogenese des Binde-
gewebes und der Muskelfasern ist identisch.

Eine quergestreifte Muskelfaser ist somit, entgegen der bis-
herigen Auffassung, keine vielkernige Riesenzelle, sondern von viel
komplizierterem Bau. Was bis jetzt Sarkolemm genannt wurde,
umschlieBt Muskelzellen, Sarkoplasma und Muskelfibrillen. Die

1) There still remain to be solved many problems involving the
voluntary muscle cells and fibres. A much greater number of lower
vertebrate muscles should be considered. Not only cardiac muscle but
unstriped fibres in the whole vertebrate series should be reviewed in the
light of the facts which I have set forth. Other pertinent questions are
the relation of the nerve fibrillae to these extra-cellular fibrillae and
several aspects of the histo-physiological phenomena of contraction; the
relation of the lymph spaces to the fibrillae; the extent of the muscle
cells in a longitudinal direction; the position of the telophragmata relative
to the muscle cells; ete., etc. Accordingly, my treatise can be considered
to be but little more than a preliminary communication.

The Relation of Muscle Cell to Muscle Fibre in Voluntary ete. 143

Zellen haben eine Membran; ihr Protoplasma besteht aus Spongio-
plasma und Hyaloplasma und umschlieft einen Kern mit Nukleolen.
Uber die Natur des Sarkolemm wird in einer folgenden Abhandlung
eingehender berichtet werden.

Bibliography.

Since the recent treatises of HEIDENHAIN and of KEIBEL and Maa
contain good working bibliographies dealing with the questions involved
in this investigation, I have considered it superfluous to append a long
literature list. In addition to some works to which I have especially
referred, I have added only those articles which have appeared since the
time of writing of these two books.

Aimr, Bandes intercalaires et bandes de contraction dans les muscles
omo-hyoidiens de la tortue. Bibliogr. Anatom., 1911.

GaGE, The Microscope. 11% Edit. Comstock Publ. oo > lthacay N.Y.

HARRISON, Uber die Entwicklung der nicht knorpelig vorgebildeten Skelett-
teile in den Flossen der Teleostier. Archiv f. mikrosk. Anat.,
Bd. XLU, 1893.

HEIDENHAIN, Plasma und Zelle. I. Lief. Handb. d. Anat. d. Mensch.
Herausg. v. K. v. BARDELEBEN, 1911.

KEIBEL und Mau, Handbuch der Entwicklungsgeschichte des Menschen.
Leipzig, S. Hirzel, 1910.

LELIEVRE et RETTERER, Des différences de structure des muscles rouges
et blancs du lapin. C. R. soc. Biologie, t. LX VI, 1909.

— —, Structure de la fibre musculaire du squelette des Vertébrés. Ibid.

LEvvie, Untersuchungen zur Anatomie und Histologie der Tiere, 1883.

Louis DES ARTS, Tiber die Muskulatur der Hirndineen. Jenaische Zeit-
Schr. i. Neate. Bd. XLIV, 1909.

Meves, Archiv f. mikr. Anat., Bd. 75, 1910.

Nussspaum, Anatomische Studien an Californischen Cirripedien. Bonn
1890, M. Cohen & Sohn.

PRENANT, Problémes cytologiques généraux soulevés par l'étude des cellules
musculaires. Journ. de l’Anat. et de la Physiol., XLVIII® Année,
1912, No. 3, Mai-Juin, p. 259.

ScHULTZE, Zeitschr. f. wissensch. Mikroskop. u. f. mikroskop. Technik,
Bd. XXVII, 1910.

Sosotra, Atlas und Lehrbuch der Histologie und mikroskopischen Anatomie
des Menschen. J. F. Lehmann, Miinchen 1911, 8S. 41.

THomA, Uber die netzformige Anordnung der quergestreiften Muskelfasern.
Arch. f. pathol. Anat., Bd. CXLI, 1908.

THULIN, Studien iitber den Zusammenhang granularer, interstitieller Zellen
mit den Muskelfasern. Muskelfasern mit spiralig angeordneten
Saulchen. Anat. Anzeiger, Bd. XX XIII, 1908.

144 W. M. Batpwiay,

Explanation of figures of Plates VII—VIIL.

Fig. I. This is an oblique section through an extrinsic muscle fibre
of the eyeball of a white mouse fourteen days old. The preparation was
stained especially deeply with Gages chloral hematoxylin. The cell mem-
brane is everywhere distinctly seen with the attached spongioplasm fibrils.
Magnification 1500 diameters.

Fig. Il. This preparation is similar to the above. The core of
protoplasm is, apparently, interrupted at a level slightly above the middle
of the section. Magnification 1500 diameters.

Fig. III. This represents a muscle cell imbedded in a thigh muscle
of an adult frog. Only the immediately adjacent muscle fibrillae are
represented. The stain used was alcoholic hematoxylin, the fibrils of
spongioplasm of the cell body, accordingly, lack the granular deposit of
the chloral hematoxylin stain used on other preparations. The outline
of the cell is most distinct. At the upper end of the sketch the internal
surface of the obliquely-sectioned cell membrane with its attached fibrils
of spongioplasm is to be observed. Magnification 1500 diameters.

Fig. IV. A preparation stained like the above and from the same
muscle. The two groups of muscle fibrillae lying above and to the left
are separated from the muscle cell by an artificial tear in the fibre. At
the left side of the cell the torn edge of the cell wall can be seen to
project into the clear space occasioned by the tear unaccompanied by
sarcoplasm. Magnification 1500 diameters,

Fig. V. A portion of a longitudinal section of a muscle fibre of
the thigh of-an adult cat stained with alcoholic hematoxylin. A slight
fold in the fibre occurring at the level of the nucleus explains the pre-
sence of the four transected muscle fibrillae. The muscle cell in this
figure is closely applied to the internal surface of the sarcolemma. Its
deeply stained protoplasm is very sharply marked of from the sarcoplasm
of the muscle fibre by the cell wall. Magnification 1500 diameters.

Fig. VI. This is a portion of a thigh muscle of an adult cat
sectioned obliquely and stained with alcoholic hematoxylin. The muscle
cell rests against the investing sarcolemma. The distinct outline of the
cell wall is not exaggerated in the sketch. The protoplasm of the cell
body is very faintly stained and stands out in sharp contrast to the uni-
formly deep stain of the sarcoplasm. This sketch was made a little above
the level of the center of the nucleus, consequently a space, occupied by
cell protoplasm, intervenes between the latter and the cell wall. At the
level of the nucleus similar cells show the nuclear membrane in contact
with the cell membrane. Upon the sarcolemma-side of the cell the wall
of the latter is fused with the sarcolemma. Magnification 1500 diameters.

Fig. VII. This is a transected extrinsic eye muscle fibre of a calf.
The stain used was alcoholic hematoxylin. The sketch was made above
the level of the center of the nucleus. The attachment of the cell wall
to the sarcolemma is well shown. The intervening area of sarcoplasm
between the cell wall and the muscle fibrillae has been carefully repre- -
sented. Magnification 1000 diameters.

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The Relation of Muscle Cell to Muscle Fibre in Voluntary etc. 145

Fig. VIII. This represents a transected fibre of the latissimus dorsi
muscle of a three-weeks old chicken. The relations are the same as those
described under fig. VII. Magnification 1000 diameters. -

Fig. IX. This is a portion of a fibre of an extrinsic eye muscle of
a calf cut longitudinally. Two muscle cells lying between the sarcolemma -
and the muscle fibrillae are represented. Notwithstanding the- marked
contrast between the structural features of the cell protoplasm and those
of the sarcoplasm, because of the overlying and parallel running fibrillae
it is impossible in this preparation to identify the cell membrane. I have
projected the telophragma lines upon the same level with the cell proto-
plasm. They do not traverse this latter structure. Stain Gages chloral
hematoxylin. Magnification 1000 diameters.

Fig. X. This is a portion of a fibre of the latissimus dorsi muscle
of a three weeks-old chicken represented in longitudinal section. The telo-
phragma lines can be seen to lie over the muscle cells. The stain used
was alcoholic hematoxylin. The cell outlines, therefore, are clearly
represented. Magnification 1000 diameters.

Fig. XI. This figure represents a longitudinal section of a fibre of
an extrinsic eye muscle of a fourteen-day old white mouse, stained with
chloral hematoxylin. Three muscle cells are seen. The general morpho-
logical appearances noted in figures IX and X are to be seen here as
well. A muscle fibre traverses the face of the nucleus on the left.
Magnification 1000 diameters.

Fig. XII. This is a muscle cell imbedded in a caudal muscle of a
tadpole about 5,0 cm long. The immediately adjacent muscle fibrillae
only are represented. At either end of the spindle-shaped cell body the
protoplasm is drawn out to a pointed extremity. The determination of
the exact extent of this protoplasm is very difficult, notwithstanding the
marked differences between its structure and that of the adjacent sarco-
plasm. The stain used was alcoholic hematoxylin and the magnification
1000 diameters.

Fig. XIII. This is a portion of a fresh thigh muscle of an adult
frog showing a muscle cell imbedded among the muscle fibrillae. The
fibre is represented in longitudinal section. The stain used was methylene ~
blue. Everywhere the cell outline stands out in sharp contrast to the
comparatively faintly stained muscle fibrillae. This specimen being a fresh
preparation and not having been treated with any fixative reagents or
other chemicals save the methylene blue stain alone represents practically
a living cell and muscle fibre in which no shrinkage, contraction or other
artefactitious change has yet appeared. The cell was located and the
sketch outlined within ten minutes from the time of removal of the
specimen of muscle from the living frog. Magnification about 1500 dia-
meters.

Nachdruck verboten.

The relation of the Sarcolemma to the Muscle Cells of voluntary
vertebrate striped Muscle Fibres and its morphological nature.

By W. M. Baldwin,
Cornell University Medical College New York City.

With plate IX.
(From the Biological Laboratory at Bonn.)
(Der Redaktion zugegangen am 1. August 1912.)

In a recent communication?) I have set forth the conception as
the result of studies upon various striated muscles that the volun-
tary striped muscle fibre of the adult vertebrate should be regarded
not as a plasmodium containing many nuclei or a giant multi-
nucleated cell, which view has been entertained up to the present
day, but, rather, as a composite contractile structure consisting of
muscle fibrillae and of sarcoplasm enclosed by sarcolemma and
containing, in addition, many muscle cells. Each one of the
numerous nuclei imbedded in the muscle fibre presents nucleoli, is
immediately surrounded by differentiated cellular protoplasm con-
sisting of strands of spongioplasm with interstices of clear hyalo-
plasm, and is completely and distinctly limited from the muscle
fibrillae and the sarcoplasm of the muscle fibre by a cell membrane.
In other words, every feature of cell structure is to be observed.
The muscle fibrillae are, accordingly, extracellular, while the sarco-
lemma should not be regarded as a cell membrane in the sense
that it envelopes a multinucleated mass of protoplasm. In my
earlier contribution, however, I left unanswered many contingent
questions, such as the structural position of the sarcolemma, its

1) Zeitschr. f, allg. Physiol., 1912.

The relation of the Sarcolemma to the Muscle Cells ecc. 147

exact relation to the enclosed muscle cells, the relation of the
telophragma lines to such cells, the longitudinal extent of the
latter, etc. The purpose of my present writing is to elucidate
certain of these problems; in particular, however, the morphological
relation of the sarcolemma to the muscle cell walls.

In accepting the claim that the muscle fibre contained eomplete
cells and not merely isolated nuclei, questions as to the relation of
the telophragmata to such cells, i. e., whether the cell protoplasm
was traversed by the uninterrupted telophragmata, were immediately
presented for solution. Granting that the telophragmata are septa
which completely traverse the muscle fibre (HemeENHAIN, CaJsaL,
Mc Catium, Renaut, Marceau, Houmaren, ete.) then at the level
of the muscle cells either the protoplasm of the latter is traversed
or the telophragmata are interrupted. In the latter contingency
especially in reference to the peripheral muscle cells the views
entertained by M. Hemennaix, Marceav, Renavut, HoumeRren,
ZIMMERMANN, that the telophragmata are directly attached to the
sarcolemma must suffer some exceptions or the sarcolemma must be
looked upon as surrounding each muscle cell in addition to investing
the fibre as a whole. Nothing, so far as I know, has been stated
definitely by the various investigators considering these problems
regarding the traversal of the differentiated protoplasm investing
the muscle nuclei. Indeed, the true nature of this protoplasm has
been, as well, overlooked. At the level of the nuclei, however, 1
seems to be generally admitted, and is well represented in many
of the figures of the workers whom I have cited, that the telo-
phragmata do not traverse the muscle nuclei.

This present contribution is based upon studies of various
voluntary muscles of Axolotl, chicken, white mouse, and cat. The
technic and staining methods employed are the same as I described:
in my preliminary contribution. The secret upon which the deri-
vation of good results depends is none other than overstaining with
long prolonged extraction. In the present study I have confined
my attention to those muscle cells which occupy a_ peripheral
position in the muscle fibre lying in contact with the sarcolemma.
The results present additional evidence bespeaking the new con-
ception: of the voluntary striped muscle fibre, which | have outlined
above, and verify with increased emphasis the fact that the muscle
fibrillae are extracellular, or intercellular, structures in the adult.

In order to present clearly and consecutively the fundamental

facts ascertained by my studies, I have selected a series of white
10*

148 W. M. BAaLpwIn,

mouse preparations which are represented in figs. 1 to 4, inclusive.
These are fibres from a thigh muscle of a three-fourths grown white
mouse and are sketched at a magnification of 1000 diameters. The
features noted in this series are to be taken not as limited to the
muscles of this form of vertebrate, but, rather, as typical of the
eeneral condition found and as well presented in all of the other
vertebrate muscles which I have studied.

In fig. 1 a portion of such a muscle fibre cut in its long axis
is represented. The free edge of the fibre presents a single muscle
cell with its relatively large nucleus. The muscle fibrillae are seen
to be deviated slightly out of their course in their passage by the
nucleus. Some of these fibrillae lie upon an optical plane lower
than the level of the nucleus. One fibril is represented as traversing
the face of the nucleus. In brief this peripheral muscle cell occu-
pied that aspect of the muscle fibre turned at right angles to the
observer. The presence of the bulgings of the sarcolemma between
the attachment of the telophragmata noted upon the right aspect
of the fibre may be accepted as evidence of the fact that the fibre
was fixed in a condition of contraction. By reason of these bulgings
the relation of the sarcolemma to the cell can be all the more
readily observed. It is seen to pass across the face of the nucleus.
Careful focusing demonstrated that the same relation obtained on
the under aspect of the cell.

The specimen was very deeply stained with chloral hematoxylin,
so deeply, in fact, that those aspects of the sarcolemma but
slightly inclined towards or away from the eye could be readily
observed as distinct laminae. Therefore it was possible to see at
the level of the serrated edge of the sarcolemma traversing the
face of the nucleus, two surfaces of sarcolemma, one upon the
uppermost optical level of the fibre and the other between this
and the muscle cell. Indeed, the continuity of these two surfaces
could be readily observed at the serrated edge. Furthermore, the
telophragmata could be followed in unbroken continuity from the
uppermost aspect to the lower around the serrated edge. Between
these two aspects lay the muscle fibril which I have represented
and to which I have previously referred. The sarcolemma was, in
fact, indented by the muscle cell and lay between the latter and
the muscle fibrillae at the level of the center of the muscle cells.
[t did not enclose the muscle cell. At the level of the center of
the muscle nucleus, because of its proximity to the nuclear membrane,
its identity was obseured.

The relation of the Sarcolemma to the Muscle Cells ecc. 149

These same relations of sarcolemma, of telophragmata, and of
muscle cell could be seen upon the under aspect of the fibre. As
1 have mentioned above, the general appearance presented was
that of an indentation of the fibre by the muscle cell.

The question naturally arising as to whether the cell under
consideration was a connective tissue cell overlying or underlying
the muscle edge is negatived by the relations of sarcolemma which
I have already outlined. Further support is contributed by a study
of the relationship of the protoplasm of the extremities of the
cell? (ec):

In such positions the serrated border of the sarcolemma occupies
the extreme edge of the muscle fibre. At such levels, too, the cell
protoplasm lies among the muscle fibrillae as I have represented,
in other words, is encompassed by the sarcolemma and the telo-
phragmata.

Fig. 2 represents a portion of a muscle fibre containing a
muscle cell similar to that in fig. 1 but rotated through an are of
90° so that the cell occupies the uppermost aspect of the fibre and
consequently faces the observer. Many of the conditions unex-
plained in the foregoing figure are readily understood by a study
of this sketch. There is no question as to its being a muscle cell
and not a connective tissue cell. It is buried in the muscle fibre. The
telophragmata are observed to mark off the bulgings of the sarco-
lemma upon either border of the muscle fibre. They do not, however,
pass across the surface of the nucleus. Herein lie the characteristic
and significant features of the specimen.

Overlying the muscle cell the serrated edge of the sarcolemma
is observed to enclose a spindle-shaped space. The telophragmata
proceed only up to this serrated edge. In this deeply-stained hema-
toxylin preparation the sarcolemma can be seen to be folded back
upon itself in order to pass around the cell body. Hence the pre-
sence of the serrated interval, hence, also, the absence of the telo-
phragmata upon the face of the cell. The sarcolemma does not
traverse this uppermost aspect of the cell. It is reflected, rather,
between the muscle fibrillae and the cell. In other words, it is
invaginated into the muscle fibre by the cell.

I assured myself that the spindle-shaped space was not owing
to the passage of the section knife through the uppermost aspect
of the fibre by a most careful study of similar chloral-hematoxylin
preparations which were especially deeply stained. In these the
sarcolemma asa distinctly stained and homogenous membrane could

150 W. M. BaLpwin,

i «

be seen to be reflected upon itself at the serrated edge. There
was no indication whatever of an artefactitious tear or section cut
in the fibre.

In fies. 3 et 4 similar muscle fibres containing peripheral muscle
cells are represented in transection. An entire muscle fibre and a
portion of another are seen in figure 3. Each contains a muscle
cell. That at A was encountered by the section knife at a level
corresponding to A in fig. 2, hence the nuclear membrane, cell
membrane, and immediately investing sarcolemma appear to be one.
The nucleus seems to be imbedded in and in direct contact with
the sarcoplasm of the fibre. Such appearances [ have commented
upon more fully in my other paper.

The cell indicated at C was transected at a level corresponding
to level C in fig. 2. Nothing of the nucleus is to be seen; strands
of spongioplasm, however, traverse the protoplasmic substance of
the cell which is invested by cell wall plus sarcolemma.

This fact must be especially noted. According to our inter-
pretation of the appearances presented by the cell in fig. 2 the
nucleus and cell wall lie in direct contact with the perimysium of
the muscle fibre, i. e., the sarcolemma does not lie upon the peri-
pheral aspect of the cell, but, on the contrary, is reflected between
it and the muscle fibrillae. The protoplasmic extremity of the cell
at C, however, does not lie upon the periphery of the fibre, but
passes deeper and deeper into the substance of the muscle fibre
lying among the fibrillae. A narrow interval of sarcoplasm, hence,
intervenes between the fused perimysium and sarcolemma encom-
passing the muscle fibre as a whole and the fused cell wall and
sarcolemma immediately investing the cell protoplasm. Just how
this sarcolemma comes to be invaginated into the muscle fibre
among the fibrillae is rendered clear by a reference to diagram 5.
Of these features I will speak more at length later.

The nucleus in fig. 4 has been sectioned at the level B (fig. 2),
i. @., through one of its poles. At this level the nuclear membrane
is separated from the cell wall and sarcolemma by an interval of
cell protoplasm. It does not lie in contact with the investing
membrane of the muscle fibre. The place of infolding of the sareo-
lemma to invest the nucleus is well represented at X. In the rest
of its extent about the muscle fibre, its identity as a distinct
membrane is lost because of its fusion with the investing peri-
mysium. The place of infolding is the serrated edge observed in
longitudinal sections (fig. 2) and encloses the cigar-shaped space

The relation of the Sarcolemma to the Muscle Cells ecc. 15k

referred to under the latter figure. Several muscle fibrillae occupy
the angles of reflection. These were observed in figs. 1 and 2 to
traverse the face of the nuclei. By reason of these infoldings the
muscle cells really lie outside of the muscle fibre. In other words,
the muscle fibrillae are intercellular. The muscle cells are merely.
received into a depression in the muscle fibre, lined with sarcolemma
while the extremities of the cells penetrate among the fibrillae in
tube-like prolongations of this same sarcolemma (c, fig. 3). The
fact should be particularly noted in this specimen, since it is well
demonstrated, that the sarcolemma is wanting between the muscle
cell and the investing perimysium of the fibre opposite the level of
the nucleus.

This interpretation of the relation of the sarcolemma to peri-
pheral muscle cells renders reasonably clear the significance of the
relation of the telophragmata to such cells. It is a significant fact
that none of the figures published represent these lines as transect-
ing the nuclei. In general they are represented as terminating
abruptly in the immediate vicinity of the nuclei. Undoubtedly,
because of too faint staining, both the infoldings of the sarcolemma
at the edges of the nuclei and the presence of the serrated edge
enclosing the cigar-shaped interval overlying the face of the nuclei
have been overlooked.

The sketch 5 is a semi-diagrammatic representation of a muscle
fibre cut longitudinally exactly through the middle of a peripheral
muscle cell. The sketch is intended to demonstrate the relation of
the sarcolemma to the cell wall. For the purpose of making this
relation clearer the cell wall, C, is drawn with a narrow interval
separating it from the sarcolemma, A—B. For the sake, also, of
clearness the cell protoplasm with its fibrillae of spongioplasm is omit-
ted. Consequently, the nucleus appears to lie in a clear space.

Tracing the sarcolemma, A, of the periphery of the fibre towards
the nucleus, we note that at F it is reflected and passes then in
contact with the cell wall, C, back to A. The angle F is the
serrated edge outlining the cigar-shaped interval mentioned above.
It contains, as shown, two muscle fibrillae cut obliquely which
diverged out of the exact long axis of the fibre in order to proceed
uninterruptedly around this interval. Between the angles of re-
flection of the sarcolemma, the cell wall lies in direct contact with
the perimysium, D. That this last named structure contains nuclei
is shown by the cell represented at E upon the left border of the
fibre. Many of such spindle-shaped cells are to be found throughout

152 W. M. BALDWIy,

the entire muscle series. But little doubt exists, then, that the
perimysium is a connective tissue structure. As yet, however, I
have failed to find a single nucleus in the true sarcolemma.

A portion of two muscle fibres cut longitudinally is represented’
in fig. 6. These are from a thigh muscle of an adult cat. By
reason of a tear.in the specimen the two fibres are separated from
each other by an interval which is bridged, nevertheless, by several
connective tissue strands. The fibre on the left contains two muscle
cells united by cellular protoplasm which tunnels the peripheral
portion of the muscle fibre. The relations of the crenated sarco-
lemma edge to the nuclei and of the perimysium to the upper muscle
cell are the same as those noted in connection with the foregoing
figures and, hence, require no further amplification. The lower of
the two cells, however, presents several elucidating features.

The crenated border of the sarcolemma passes across the face
of this nucleus. Because of the separation of the two fibres, however,
the fibrillae constituting the perimysium are pulled away from the
cell body and from the lower portion of the muscle fibre. One of
these fibrillae is seen to be derived from a connective-tissue cell.
Hence, the lower portion of the muscle fibre is naked so far as its
perimysium investment is concerned. The sarcolemma alone clothes
this portion of the fibre, and in it, incidentally, no cells are to be
found. The removal of the perimysium has left the cell wall bare.
The delicate nature of this structure, therefore, can be seen. In
the extreme lower end of the sketch the cell protoplasm is observed
to pass into the interior of the muscle fibre. Careful focusing and
a study of the relation of the telophragmata to this protoplasmic
mass proved that this cell extremity lay imbedded among the
fibrillae of the muscle fibre. This specimen furnishes the most
conclusive evidence upon the identity of the sarcolemma as a
homogenous membrane, of the perimysium as a connective tissue
structure, and of the presence of a distinct cell wall of the muscle
cells.

The following four sketches are presented for the purpose of
demonstrating the intimacy of contact of the sarcolemma with the
perimysium. The first two figures are precisely similar to such
transections which are ordinarily represented in text-books. The
sketch (fig. 7) was taken from a section of thigh muscle of a white
mouse three-fourths grown and demonstrates portions of four muscle
fibres transected. The nucleus at A is that of a peripheral muscle
cell that at B, a perimysium cell. The latter possesses a spindle-

The relation of the Sarcolemma to the Muscle Cells ecc. 153

shaped body from whose extremities the perimysium passes off to
encircle the muscle fibre.

Fig. 8 is a portion of a transected thigh muscle of an adult
eat. The cell at A is a connective tissue cell and gives off several
processes. The cell at B is similar to the cell indicated at B in -
the foregoing figure, a spindle-shaped perimysium cell whose pro-
cesses are incorporated in that structure. In the instance of both
of these perimysium cells the observation can very readily be made
that, apparently, at least, the perimysium splits at the cell to en-
close it, yet the internal of the two layers formed by this splitting
process is so thin that, opposite the middle of the cell nucleus, its
presence cannot be determined. Indeed, between the nucleus and
the sarcoplasm no formed structures can be discerned, yet our
studies of longitudinal sections, as we have seen above, show that
the exceedingly thin sarcolemma does lie between the fibrillar, cell-
containing perimysium and the sarcoplasm. Its thinness and its
intimacy of contact with the perimysium are such that, only in
those places where it leaves the latter to be reflected around the
peripheral muscle cells, can its identity be recognized.

That this intimacy of contact with the perimysium is real and
not apparent, the last two sketches demonstrate. The muscle fibre
in fig. 9 is a transected trunk muscle of Axolotl and that in fig. 10
a similar section of the latissimus dorsi of a three-weeks old chicken.
In both specimens, tears had been caused. As a result the peri-
mysium was pulled away from the sarcoplasm. It is to be noted
that the sarcolemma adheres to the former. The evidence for this
fact must be given negatively, since is not demonstrable upon the
border of the sarcoplasm imbedding the muscle fibrillae. This is
seen to be rather ragged in outline such as would not be expected
were the sarcolemma in situ. The appearances in fig. 10 seem to
negative this latter statement, however, since the free edge of the
sarcoplasm appears to possess a sharply defined thickened border,
— a true sarcolemma. It must be noted in the sketch, however,
that a small lappet upon the left aspect of the sarcoplasm indicates
that a tear through the latter had been occasioned in the specimen.
As a result a group of thirteen or fourteen fibrillae with their im-
bedding sarcoplasm was pulled away from the main fibril group.
The significant fact demonstrated by the specimen is that in the
angle of this tear the same apparently sharp edge of the sarco-
plasm is observed. In other words, the edges of the torn sarco-
plasm present the same distinct border as is observable elsewhere

154 W. M. BaLpwiy,

upon the surface of the sarcoplasm. Therefore, it is very probable
that this seeming border is not sarcolemma but an artefactitious
product, a kind of superficial increase in density of the sarcoplasm.

The remarks which I have made above and the conclusions
which I have drawn, apply particularly to peripheral muscle cells.
[ feel sure by induction, however, that the same relation of sarco-
lemma to cell wall and of cell wall to cell protoplasm obtains in
the instance of central-lying muscle cells. In fact, 1 have observed
many such cells encompassed by sarcolemma. Until a new nega-
tive staining method is devised by which the cell protoplasm is
stained and the muscle fibrillae unstained, however, I cannot commit
myself upon the derivation of the sarcolemma immediately investing
the cell wall, i. e., whether it represents a portion isolated from
that enveloping the fibre or is derived from the same in a tube-
like process tunneling the length of the muscle fibre. So far as I
have gone with my research studies the latter appears to be the
case, yet the evidence is not so coherent as to be presented in an
absolutely convincing whole, by reason, as I have mentioned above,
of a staining difficulty. In brief, the fundamental problem involved
is the determination of the exact extent of the protoplasmic masses
belonging to the imbedded cells. The difficulties present in the
solution of this problem still await solution.

I have not yet observed a single centrally-located muscle cell,
however, whose protoplasm was traversed by the telophragmata of
the muscle fibre. I am in a position further to corroborate fully
the views entertained by HerpEnHAIN, SCHIEFFERDECKER, Mc CaLLum,
Enpertery, Houmeren, Zimmermann, among others, that in the
adult striped muscle the telophragmata are directly attached to
the sarcolemma. The only interruptions occurring in this attach-
ment are apparent rather than real and are to be noted in con-
nection with the protoplasmic masses of the central and of the
peripheral muscle cells. In such instances the telophragmata are
directly attached to the sarcolemma immediately investing these
muscle cells. They do not traverse either the protoplasm or the
nucleus of such cells. Such observations as those of ENDERLEIN on
Gastrus equi and on Hypoderma Diana, where the telophragmata
traversed the protoplasmic cellular masses outlying the muscle
fibrillae but enclosed within the sarcolemma, I have failed to verify
in the adult muscles of the higher vertebrates which I have studied

Without entering too extensively into the history of the investi-
cations upon the nature and relations of the sarcolemma, the following

~

The relation of the Sarcolemma to the Muscle Cells ecc. 15)

brief statements of the more pertinent facts should be made. As
early as 1840 Bowman named the structure, which we have generally
considered as sarcolemma, and described it as a structureless mem-
brane serving as an envelope for the muscle fibres. RercHErtT sub-
sequently questioned this view of its morphological character, however,
Maintaining that it was a connective tissue structure and contained
nuclei. These two papers marked the beginning of a half-century
dispute which has lasted down to the present day. The credit
belongs to Leypic, however, for first advancing the view of the
sarcolemma of the vertebrate muscle as a cuticular structure.
CaLBERLA also held the same opinion. Hentz, on the contrary,
found nuclei in it. Casat, Hocur, Hemernnary, Marceau, Renavt,
ZIMMERMANN, and HoumGren concluded, in spite of the fact that
they could discerm fibrils and cells in the sarcolemma, that in
cardiac muscle fibres the latter should be regarded as a cuticular
thickening of the sarcoplasm. One of the most recent contributors
upon the subject is SCHIEFFERDECKER. This investigator utilized
the trunk musculatur of Petromyzon for his purposes. In the
“central” fibres of such muscles he found the examples, long desired
by investigators, where sarcolemma, devoid of perimysium, alone
enveloped the fibres. In the study of such specimens he came to
the conclusion that the sarcolemma of the voluntary striated muscle
was a homogenous cuticular thickening and possessed neither cells
nor fibrils. I, too, have studied such specimens and, as well, have
arrived at the same conclusions. I differ in opinion from the latter
and from Mavrer, among others, however, in regarding this thick-
ened cuticula not as a cell membrane. It is apparently a deriva-
tive of the sarcoplasm, by a process of condensation and in this
respect alone resembles a cell membrane. It does not, however,
encompass a multinucleated mass of protoplasm, since the sarco-
plasm imbedding the muscle fibrillae, as I have demonstrated above
does not contain nuclei.

My own studies have demonstrated in addition, that in the
muscles which I have utilized and under the conditions which I
have enumerated the sarcolemma of the adult, striped, voluntary
muscle can be observed as a homogenous, fibre-less, and cell-less
membrane.

The cause of the long controversy upon the subject is referable
to the fact that the structure seen in ordinary transections ot
voluntary striped muscle fibres, as figures 7 and 8 show, is not sarco-

156 W. M. BaLpwihy,

lemma but sarcolemma plus perimysium. The cells and fibres de-
tected in it belong not to the sarcolemma but to the perimysium.

The conclusions to which I have arrived, then, as the result of
my studies upon these adult voluntary striated vertebrate muscles are:

The sarcolemma is a thin, structure-less, homogeneous mem-
brane completely investing the muscle fibre.

2. It contains no cells and no fibrillae.

3. It is infolded from the perimysium to invest the peripheral
muscle cells.

4, At such places it separates the muscle cells from the sarco-
plasm of the muscle fibre.

5. At such places, likewise, the muscle cell wall opposite the
level of the nucleus lies in direct contact with the perimysium.

6. Upon its internal surface the sarcolemma is always in direct
contact with the sarcoplasm.

7. The telophragmata are always directly attached to its inter-
nal Cee

8. Its external surface is fused with the perimysium.

9. The sarcolemma and the perimysium together constitute the
investing envelope of the muscle fibre.

10. Only at the position of the peripheral muscle cells are
these two structures separated from each other.

11. The perimysium is a connective tissue structure containing
both spindle-shaped cells and fibres.

12. The sarcolemma is so thin and so closely applied to the
perimysium as to escape observation in transections excepting at
those levels where it is infolded to invest the peripheral muscle
cells.

13. It can be seen to best advantage as a separate layer in
chloral-hematoxylin preparations and at those levels in longitudinal
sections where it is infolded to invest the peripheral muscle cells.

14. The telophragmata do not traverse the protoplasm of the
muscle cells nor their nuclei.

All of these results are merely confirmatory of my earlier ob-
servations, i. e., — that the voluntary, striped muscle fibre of the
adult vertebrate is not a giant, multi-nucleated cell, but, on the
contrary, a composite contractile structure consisting of muscle
fibrillae and of sarcoplasm enveloped by sarcolemma, and, in
addition, numerous muscle cells. In other words, the muscle fibrillae
are extracellular structures. The muscle nuclei are not directly
imbedded in the sarcoplasm of the fibre but are separated from it

The relation of the Sarcolemma to the Muscle Cells ecc. 157

by a layer of sarcolemma and by cell protoplasm which possesses
a distinct cell wall. The terms muscle fibre and muscle cell as
applied to this form of striped muscle are not synonymous. Further-
more, the muscle fibrillae are comparable to the fibrillae of adult -
connective tissues to the extent, at least, that both are intereellular
in position.

Zusammenfassung.

Das Sarkolemm ist eine diinne, strukturlose, homogene Hiille
der Muskelfaser ohne Zellen und Fibrillen. Seine iuSere Oberfliche
schmiegt sich dem Perimysium an und nur da, wo die Muskelzellen
gelegen sind, weicht es von dem Perimysium zuriick, so da die
Muskelzellen zwischen Perimysium und Sarkolemma liegen und das
Sarkolemm nur das Sarkoplasm und die Muskelfibrillen umschlieBt.

Die Telophragmata sind mit der inneren Oberfliche des Sarko-
lemms verwachsen und dringen nicht in das Protoplasma der peri-
pheren Muskelzellen ein.

Das Perimysium ist bindegewebiger Natur: es enthilt Fasern
und spindelférmige Zellen.

Perimysium und Sarkolemm zusammen umgeben die Muskel-
faser und sind nur dort voneinander getrennt, wo die peripheren
Muskelzellen gefunden werden. Das Sarkolemm ist so diinn, daf es
an Schnitten der Beobachtung sich entzieht, aufer an den Stellen,
wo es in der Gegend der peripheren Muskelzellen yon dem Peri-
mysium sich zuriickklappt und eine Mulde fiir diese Zellen bildet.
Am besten kann das Sarkolemm an Chloral-Haimatoxylinpriparaten
sichtbar gemacht werden.

Somit sind die Muskelfibrillen extrazelluliire Bildungen. Die
Muskelzellen sind yom Sarkoplasma und den Muskelfibrillen durch
das Sarkolemm getrennt und gemeinschaftlich mit diesen beiden
Bestandteilen der Muskelfaser yom Perimysium umgeben. Die
Muskelzellen haben eine deutliche Zellmembran. Die Muskelfibrillen
sind den ausgebildeten Bindegewebsfibrillen vergleichbar, da beide
extrazellular gelegen sind.

Muskelfaser und Muskelzelle sind nicht synonym, soweit es sich
um die hier untersuchten Arten quergestreifter Muskeln handelt.

5R W. M. BALDWIN,

Bibliography.

Bowman. Phil. Transactions, Vol. 2, 1840, Vol. I, 1841.

CALBERLA, Archiv f. mikr. Anat., Bd. XI.

Casa, Intern. Monatschr. 5. Bd., 1888.

ENDERLEIN, Archiv f. mikr. Anat., 55 Bd., 1900.

HerrpENHAIN, Plasma u. Zelle in Handbuch d. Anat. d. Menschen, hrsg.
yon v. Bardeleben, 1911.

HeNuE, Allgemeine Anatomie, 1841.

Hocueg, Bibliogr. Anat., No. 3, 1897.

HormGREN, Anat. Anz., 31. Bd., Archiv f. mikr. Anat., 71. Bd.

LeypiG, Untersuchungen zur Anat. u. Hist. der Tiere, 1883.

McCauuum, Anat. Anzeiger, 13. Bd., 1897.

Marceau, Bibliogr. Anatomique, 1902. — Amn. des Sc. nat., 8 série,
Zoologie, T. 19, 1903. — Arch. d. Anat. miecr., T. 7, 1905.

Maurer, Handb. d. Entwicklungsl., hrsg. von O. Hertwig, 3. Bd., 1. Teil,
1906.

ReIcHERT, Archiv f. Anat. u. Phys., 1841.

Renavut et Mouuarp, J., Revue génér. d. hist. T. I, Fase. 2, 1905.

SCHIEFFERDECKER, Untersuchungen iiber die Rumpfmuskulatur von
Petromyzon fluviatilis, etc. Archiv f. mikr. Anat., Bd. 78, 1911.

ZIMMERMANN, Arch. f. mikr. Anat., 75. Bd., 1910.

Explanation of figures of Plate IX.

Fig. 1. A longitudinal section of a portion of a thigh muscle of a
three-fourths grown white mouse. A peripheral muscle cell with its
prominent nucleus is located upon one side of the fibre. A indicates the
level of the center of the nucleus; B the extremity of the same where
the nuclear membrane lies at some distance from the cell wall; C the
protoplasmic extremity of the cell prolonged into the muscle fibre among
the fibrillae. Magnification 1000 diameters.

Fig. 2. A surface view of a peripheral muscle cell of a muscle fibre
the same as that in fig. 1. The serrated edges of the reflected sarco-
lemma traverse the face of the nucleus leaving a space between in which
the muscle cell wall lies in direct contact with the perimysium. The
letters A, B, and C correspond to those of fig. 1. Magnification 1000

diameters.

The relation of the Sarcolemma to the Muscle Cells ecc. 159

Fig. 3. <A transection of two muscle fibres derived from the same
source as that of fig. 1. At A a peripheral muscle cell is represented
cut at the level indicated by the same letter in figs. 1 and 2. The
nucleus lies in direct contact with the cell wall. At C another peripheral
muscle is seen. This was encountered at the level C of figs. 1 and 2,
i. e., only the protoplasm of the cell prolongation is seen. A layer of
sarcolemma invests the cell wall. A narrow interval of sarcoplasm inter--
venes between this and the investing sarcolemma and perimysium of the
muscle fibre, P. Magnification 1000 diameters.

Fig. 4. Similar to fig. 3. At B a muscle cell cut transversely at
the level B of figs. 1 and 2 is seen. A narrow interval of protoplasm
intervenes between the nucleus and the cell wall. At x the sarcolemma
is reflected from its position in contact with the perimysium to invest the
muscle cell, thereby separating it from the sarcoplasm and fibrillae of
the muscle fibre. Magnification 1000 diameters.

Fig. 5. A semidiagrammatic sketch of a surface view of a portion,
of an adult voluntary striped muscle fibre cut through the median longi-
tudinal axis of a peripheral muscle cell. The cell wall, C, sarcolemma
A—B, and perimysium, D, are separated from each other by a narrow
interval. The protoplasm of the cell is omitted. FF, the angle of infolding
of the sarcolemma by which is produced the serrated edge noted in fig. 2
as traversing the face of the nucleus. Between the two angles of reflection
(upper and lower in the figure) the cell wall lies in immediate contact
with the perimysium. At E a spindle-shaped cell of the perimysium is
shown. Magnification about 1500 diameters.

Fig. 6. A portion of two adjacent thigh muscle fibres of an adult
cat cut in their long axis. Between the two fibres the interval, occasioned
by handling of the tissue, is traversed by several strands of perimysium
fibrils. At the lower end of this interval a connective tissue cell receives
one of these fibrils. Two peripheral muscle cells occupy the right border
of the muscle fibre and are connected with each other by a strand of
protoplasm. The serrated edge of the sarcolemma traversing the face of
the nuclei is well shown. At D the perimysium is torn away from the
muscle fibre leaving the cell membrane at C bare. Magnification 1000
diameters.

Fig. 7. In this sketch a portion of four muscle fibres of a thigh
muscle of a three-fourths grown white mouse is shown. The fibres are
transected and demonstrate two nuclei, the one on the left being that of
a peripheral muscle cell cut at the level A of figs. 1 and 2 and for this
reason not demonstrating either the cell wall or the sarcolemma inter-
vening between it and the sarcoplasm, while that on the right is a spindle-
shaped perimysium cell. Between this latter cell and the enclosed sarco-
plasm of the fibre the sarcolemma cannot be made out by reason of its
thinness and the closeness of application to the perimysium. Magnification
1500 diameters.

160 W.M. Baupwrn, The relation of the Sarcolemma to the Muscle ecc.

Fig. 8. This represents a portion of a transected muscle of the
thich of an adult cat. A connective tissue cell with several fibres is
adjacent to the perimysium which presents a spindle-shaped cell. Between the
latter and the sarcoplasm the sarcolemma cannot be seen as a distinct layer.

Fig. 9. This figure is presented to show that, in a transected
voluntary muscle of Axolotl where the investing envelope has been torn
away by mechanical means from the fibrillae, the adhesion between the
sarcolemma and the perimysium is greater than that between the former
and the sarcoplasm. A bipolar perimysium cell is seen in the torn,
envelope. The sarcolemma, exceedingly thin and though not demonstrable
upon the internal surface of the perimysium because of its intimate
incorporation with the latter, is very manifestiy not present upon the
ragged, and irregular edge of the sarcoplasm. Magnification 1000 diameters.

Fig. 10. A transected fibre of the latissimus dorsi muscle of a
three-weeks old chicken. The features demonstrated are similar to those
of fig. 9. Magnification 1500 diameters.

> oe

hifi. fiir allgem. Physiologie Bd. XIV.

Fig. 6.

Fig. 5.

Verlag v

W. M. Baldwin del.

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in Jena.

(From the Biological Laboratory at Bonn.)

The Relation of Muscle Fibrillae to Tendon Fibrillae
in voluntary striped Muscles of Vertebrates.
By
W. M. Baidwin

(Dept. of Anatomy, Cornell University Medical College, New York City.)

With plate VII.

e

The long-contended question over the structural relationship
of tendon fibrillae to muscle fibrillae has been brought to our atten-
tion by a recent contribution of O. Scuutrze. This investigator
carried out his studies on various muscles of Hippocampus, Amphi-
oxus, several amphibia, and man, and arrived at the conclusion
that these two structural features, muscle fibril and tendon fibril,
were directly continuous with each other, perforating the sarco-
lemma. Being at the time engaged in a study of certain morpho-
logical features of striped-muscle structure, I reviewed my own
preparations demonstrating these features with the express purpose
of advancing our knowledge in this subject over a larger number
of higher vertebrates than this author had used.

The preparations comprised sections of various muscles, such
as intercostaies, latissimus dorsi, rectus abdominalis, gastrocnemius,
erector spinae, extrinsic muscles of the eyeball and various muscles
of the thigh (together with caudal muscles) of such vertebrates as
the tadpole, frog, calf, cat, white mouse, chicken, gray mouse. In
addition I utilized specimens of living muscle of the frog and of
the tadpole as controls to the fixed and stained preparations. The
paraffin method of imbedding was employed in conjunction with
ScHULTZkE’s excellent collodion and chloroform method of infiltration.
The sections varied in thickness from 2 u to 5 wu.

The stains used were picric acid, methylene blue, fuchsin S, and
eosin, together with combinations of these and various alcoholic

Morpholog. Jahrbuch. 45. 16

250 W. M. Baldwin

and aqueous solutions of hematoxylin among the latter ScHuULTZE’s
and also GAGE’s. Several of the more significant of the preparations
were stained, decolorized, and then restained by another method
in order not only to serve as controls to the simple stained sections
but to demonstrate, in addition, the reaction of the several struc-
tures under consideration to the various methods previously employed.
By means of a method of blunt dissection of the fixed and stained
preparations upon the slide, several muscle fibres with their tendons
were isolated from adjacent structures, by which was rendered
possible a more careful and detailed study of their structural
relationship.

In fig. 1 I have represented such an isolated muscle fibre with
its attached tendon of an extrinsic eye muscle of a three-weeks old
chicken. The instance is typical of the usual fibre-termination ob-
served in these muscles. It can be seen that the sarcolemma at
its extremity is drawn out to three distinct pointed processes which
are not in airy way continuous either with the sheath of the tendon
or with the fibrillar components of the tendon. Furthermore, the
pointed extremities of the tendon fibrillae are observed to be inserted
into the recesses between the sarcolemma processes. This feature
is worthy of special attention because owing to it an appearance is
produced, when such an arrangement is rotated and studied in a
vertical optical plane, of tendon fibrillae lying inside of the sarco-
lemma sheath of the muscle fibre. The individual muscle fibrillae,
upon approaching the sarcolemma, lose their several features of
cross-striation but can be traced, however, as slender, faintly-stained
thread-like structures up to the internal surface of the sarcolemma
upon which they terminate. The sarcolemma is in general much
thinner than the cross-diameter of an average muscle fibril. Hence
from the morphological arrangement of the parts thus effected, the
muscle fibre appears to be dovetailed into the tendon with this ex-
ceedingly thin membrane as the only structure Sep araeee the muscle
fibrillae from the tendon fibrillae.

Fig. 2 is another fibre from the same muscle, and demonstrates
the same general features. Two muscle nuclei imbedded in granular
protoplasm separate two groups of muscle fibrillae from each other.
Kach group, however, terminates in several pointed sarcolemma
processes which are dovetailed with the fibrils of the corresponding
tendon-fibril groups. Here again the muscle fibrillae, losing gradu-
ally their features of cross-striation, are, nevertheless, readily trace-

The Relation of Muscle Fibrillae to Tendon Fibrillae ete. 251

able up to the internal surface of the sarcolemma. There is no
evidence, however, either upon morphological or upon staining-
reaction grounds for the assumption that the fibrillae of muscle and
tendon penetrate the sarcolemma. Neither can it be demonstrated
that the sarcolemma is prolonged over the tendon or among its con-
stituent fibril bundles. It terminates bluntly, rather, in a number
of cone-shaped processes.

Fig. 3 represents the termination of an extrinsic muscle of the
eyeball of a twenty-two-day-old white mouse. The observations
made above in connection with the chicken muscles apply equally
well to the muscles of this group in the mouse. Again, there is
observed, the dovetailing of the sarcolemma processes with the
tendon fibrillae, the termination of the muscle fibrillae at the sarco-
lemma, and the separation by the latter of the tendon fibrillae from
the former.

I particularly desire to refer again to the insertion of the tendon
fibrillae into the intervals between the cone-shaped prolongations
of the sarcolemma through which arrangement certain appearances
are produced which are exceedingly liable to be misinterpreted. This
thin membrane is the only structure separating the muscle fibrillae
from the tendon fibrillae. The latter pursue a course parallel to
that of the former but terminate bluntly upon the external surface
of the sarcolemma, whereas the muscle fibrillae run for a com-
paratively short distance upon the internal surface of the sarco-
lemma and finally lose their identity by fusing with it. Such con-
clusions can only be drawn from a study of those fibrillae which
lie upon the same horizontal optical plane, i. e., upon that aspect of
the sarcolemma which faces at right angle to the observer. When
the muscle fibres and tendon fibres are cut in exactly their long
axis, one must bear in mind that the uppermost and undermost
aspects of the cone-shaped sarcolemma end are obliquely inclined
to the vertical optical axis of the observer. This fact adds to the
difficulty of interpreting the relation of the fibrillae more so than
would be the case if the sarcolemma surface lay in a horizontal
optical plane. Hence the solution of the question is much dependent
upon the manipulation of the fine adjustment screw. Bearing in
mind the fact that the sarcolemma is so thin as to be almost per-
fectly transparent, when studied upon these aspects of the fibres,
the difficulty in determining the exact relationship of the muscle
fibrillae to the overlying or underlying and parallel-running tendon

16*

252 W. M. Baldwin

fibrillae is well nigh impossible with our present optical instruments.
When viewed in such vertical planes it appears as if those tendon
fibrillae terminating in the sarcolemma indentations were really
within the muscle fibre. Undoubtedly a neglect to take into con-
sideration these facts has led to several erroneous conclusions such
as are represented in various published figures. Aceordingly, I have
preferred to base my conclusions upon a careful study of those fibrillae
which occupy the same horizontal optical plane. Under such cireum-
stances the sarcolemma presents an unbroken contour. No evidence
can be found bespeaking a continuity of tendon fibril with muscle
fibril among these various extrinsic eye muscles of the white mouse,
gray mouse, chicken, or calf.

One of the best bits of evidence upon this question is furnished
by muscles of the bipenniform type. I have represented in fig. 4
a portion of a single muscle fibre of this type of an adult white
mouse with its attached central tendon. As is readily seen the
tendon fibrillae (A) lie at an angle of about 125° with the muscle
fibrillae (C). The sarcolemma investing the fibre is considerably
thickened at that end applied to the tendon. The numerous muscle
fibrillae are represented half-schematically but their relation to this
sarcolemma end is faithfully reproduced. Between the sarcolemma
and the tendon there are to be seen several layers of connective
tissue fibres and cells (6). I was able by means of various methods
of hematoxylin staining to stain at once these three structures,
central tendon, intervening connective tissue, and muscle fibrillae
three different colors upon the same slide. The tendon fibrillae
were yellowish-white, the connective tissue (peritendinum) reddish-
brown, and the muscle fibrillae deep brown. There was absolutely
no structural continuity to be seen between either the tendon fibrillae
and the connective tissue fibrillae or between the latter and the
muscle fibrillae. At no place could it be demonstrated that the muscle
fibrillae traversed the connective tissue sheath in order to reach
the central tendon, nor did they perforate the sarcolemma and turn
at an angle to join the connective tissue fibrillae.

In muscles belonging to this type of structure the relation of
the muscle fibres to the tendon is precisely similar to that of those
muscles which are attached to bones, to the bone upon which they
find their insertion. Such muscle fibres have only an indirect rela-
tion to the bone since their actual attachment is direct to the peri-
osteum. Such is the case with these bipenniform muscles. Only

The Relation of Muscle Fibrillae to Tendon Fibrillae ete. 253

through the medium of this tendon-investing, connective tissue sheath,
or peritendinum, comparable to the periosteum in the above instance,
do the muscle fibres establish their connection with the tendon.
The matter involved then is a consideration rather of the relation
of the muscle fibrillae to the peritendinum (peritenonium) fibrillae.

A similar instance of a musele fibre of this type with its attached
tendon is represented in fig. 5 under a magnification of 1500 dia-
meters. The peritendinum, consisting of several layers of connective
tissue fibrés and cells, separates the obliquely-inclined muscle fibre
from the tendon. Due to a tear in the tissues an interval exists
between a portion of the muscle fibre and the peritendinum and
another between the latter and the tendon. In the rest of their
extent however, the various structures have maintained their proper
relationship to each other. The figure demonstrates five muscle
fibrillae (A), which proceed directly up to the sarcolemma without
losing their features of cross-striation, such as was the case ob-
served with the mouse and chicken extrinsic eye muscles. The
tendon end of the sarcolemma is very noticeably thickened and
presents upon its internal surface several small elevations upon each
of which a muscle fibril is inserted. Sections stained with picro-
fuchsin demonstrate that such elevations belong to the sarcolemma
rather than to the muscle fibrillae. Moreover, sections stained with
hematoxylin-fuchsin indicate that the sarcolemma, with these ele-
vations has a different staining reaction from that of the peritendinum,
and that both of these differ from the tendon. Morphologically there
is no evidence that the sarcolemma is any other than a homo-
geneous structure unperforated and untraversed by any formed
fibrillae. With differential staining it appears as a thickened homo-
geneous, unstriated, and non-fibrillar membrane. Nor is there any
evidence of the peritendinum fibrillae turning at an angle to per-
forate it.

Another instance of the general type of muscle termination as
demonstrated in figs. 1, 2, and 3 is furnished by the caudal muscu-
lature of the tadpole. A portion of a muscle fibre with its attached
tendon fibrillae is represented in fig. 6. This fibre was removed
from a tadpole about 5,0em long. The sarcolemma is very thin
and is seen in the figure to be drawn out into a number of cone-
shaped processes. Into each of these prolongations as many as
from ten to forty muscle fibrillae enter and, without suffering any
reduction in diameter or losing their features of cross-striation,

254 W. M. Baldwin

proceed directly up to the internal surface of the sarcolemma with
which they fuse. To each one of these cone-shaped sarcolemma .
processes a single tendon fibril is attached. These fibrillae vary
among themselves in diameter, the average size is, however, about
that of a muscle fibril, which, on the contrary, are generally uni-
form in size. Were the muscle fibrillae in direct continuity with
the tendon fibrillae then each one of the former must be reduced
ereatly in diameter before becoming continuous with a tendon fibril.
There is, however, no morphological evidence of such a reduction
in size.

Unlike the tendon end of the sarcolemma in such muscles as
are of the bipennitorm type, the sarcolemma of these cone-shaped
processes is not noticeably thickened. On the contrary, it is re-
markable for its uniform thinness. Were it thickened, one might
look therein for morphological evidence of muscle fibrillae, reduced
in calibre, passing along its surface or through its substance in
order to establish a conjunction with the single tendon fibril.

I have already stated that the tendon fibrillae vary in size.
Such variations are not always proportionate, however, to the varia-
tions in size of the sarcolemma processes to which they are attached
or to the number of muscle fibrillae therein contained. Such might
be the case were a direct continuity of the two structures present.
The absence of a correlation in size among these several structures
might be adduced, therefore, as an additional fact arguing against
the continuity of the tendon fibriliae with the muscle fibrillae.

In the figure 6 two cells are demonstrated among the tendon
fibrillae. Judging from their morphological appearances and their
relation to the tendon fibrillae, I have concluded that such cells
were fibroblasts. In some instances I have found the tendon fibrillae
traversing the cell protoplasm. In the figure the larger of the two
cells gives off two delicate fibrillae each of which is attached to a
pointed extremity of the sarcolemma. ‘These are, moreover, the
only fibrillae attached to these respective sarcolemma extremities.
Upon the other side of the fibroblast several similarly slender fibrils
stream off in the general direction of the other tendon fibrillae. - 1
have, naturally, interpreted such fibroblastic processes as developing
tendon fibrillae, and upon this interpretation have found an ex-
planation for the disparity in size between the several fibrillae i. e.
the smaller tendon fibrillae represent younger fibrillae. Hence the
size of such fibrillae is in no direct wise associated with the size

The Relation of Muscle Fibrillae to Tendon Fibrillae ete. 955

of the cone-shaped sarcolemma processes upon which they are inserted
nor with the number of muscle fibrillae which terminate in such
sarcolemma processes.

Apart from these considerations, however, the staining reactions
demonstrate very clearly that the tendon fibrillae pass only up to
the sarcolemma. They do not penetrate it, neither do the muscle
fibrillae. The thickness of the sarcolemma everywhere separates
the two structures from each other.

Further evidence is contributed by the developing intercostal
muscles which I have studied, and of which I have sketched three
fibres in figs. 7, 8, and 9. In figure 7 each of the three pointed
extremities of the sarcolemma has a connective tissue fibril attached
to its apex. The fibril on the right is seen to be derived from a
distinct cell body which encloses a relatively large nucleus. The
elements of cross-striation do not accompany the muscle fibrillae up
to the extremity of the sarcolemma. In the vicinity of the latter
these fibrillae somewhat resemble the tendon fibrillae in morpho-
logical characters, yet there is no morphological appearance to be
noted from which it might be inferred that the homogene®Bus sarco-
lemma is perforated by the passage of either form of fibril. The
staining of the section shows, in addition, that whereas the muscle
fibrillae are thin and very faintly stained, the tendon fibrillae in
these instances are relatively deeply stained and much thicker. At
no place have I seen these tendon fibrillae inside of the sarcolemma
as SCHULTZE has represented in his figures.

Fig. 9 represents somewhat semidiagrammatically the typical
muscle termination observed in these developing muscles. The
termination of the muscle fibre as a whole is seen to be bluntly
pointed, but the sarcolemma end is observed, in addition, to present
upon close examination with a high power very many small cone-
shaped processes to each of which a connective tissue fibril is at-
tached. The younger the muscle fibre, the smaller are these pro-
cesses and the less the numbre of muscle fibrillae terminating within
each of them. In the course of development as these processes
increase in size and the number of contained muscle fibrillae par-
takes of a proportional increment, but a single tendon fibril, not-
withstanding, is found to be attached to the apex of these processes,
in other words these apical fibrillae do not multiply in this type
of muscle. Herein lies another significant fact denying the con-
tinuity of tendon fibrillae with muscle fibrillae.

256 W. M. Baldwin

Fig. 8 is of especial significance because it represents in the
same muscle fibre the two forms of muscle ending which I have
mentioned above, the one in which the fibre is inserted upon on
obliquely-placed surface, as represented by the instance of the bi-
penniform muscles sketched in figs. 4 and 5,-and the other where
the insertion takes place upon a tendon extremity whose fibrillae
pursue the same linear direction as those of the muscle fibrillae.
At the same time this particular fibre affords an explanation of the
genesis of the sarcolemma eminences or projections to which I
have previously referred and figured in sketches 4 and 10 (A), which
occur upon the internal surface of the tendon end of the sarco-
lemma and afford attachment to the muscle fibrillae. The right side
of this sketch demonstrates three cone-shaped sarcolemma prolonga-
tions to each of which a tendon fibril is attached. These fibrils
are typical of the arrangement observed in the tadpole tail. That
portion of the sarcolemma end upon the left side of the figure,
however, is closely applied to the obliquely-placed fibrillae of the
perichondrium enclosing the cartilage of the developing rib. The
component structures upon this side of the muscle fibre have the
same arrangement as was seen in the bipenniform muscles. The
arrangement upon the right side is to be regarded accordingly as
transitional, since in the adult animal all of the muscle fibrillae
are disposed as is shown upon the left side of the figure. This
single muscle fibre, then, represents at once the earlier develop-
mental condition and as well the adult condition; therefore, there
can be found in it the probable explanation of the origin of those
projections of the sarcolemma which are shown in fig. 10 (A).

In the earlier developmental stages the muscle fibres terminate
in the manner represented in fig. 9 i. e., by a number of cone-
shaped sarcolemma processes to each of which a connective tissue
fibril is attached. In the course of development as the muscle fibres
gradually approach and are applied to their definitive insertion, in
such instances where this insertion is upon an obliquely inclined
structure, as periosteum or peritendinum, the sarcolemma loses these
cone-shaped terminal features. This takes place by a flattening of
the apices of these cones and a synchronous fusion of the adjacent
walls of neighboring cones. By the flattening of the terminal sarco-
lemma a better adhesive surface is presented to the flat surface
of the structure affording attachment to the muscle. By the fusion
of the adjacent cone-walls is brought about the presence in the

The Relation of Muscle Fibrillae to Tendon Fibrillae ete. 257

adult of the projections of the sarcolemma which extend into the
muscle fibre. They have no connection developmentally or morpho-
logically with the tendon fibrillae, since at first there are no tendon
fibrillae attached to that portion of the sarcolemma from which
these projections are derived. Neither is it probable that they re-
present a portion of the muscle fibrillae which might have become
transformed into sarcolemma-like tissue.

This genetic sequence explains as well the appearances presented
by such muscles as I have represented in the first three figures
where the tendon fibrillae and the muscle fibrillae occupy the same
linear direction and where in the adult condition the several pointed
extremities of the sarcolemma, characteristic of the younger develop-
mental stages, are still demonstrable. The tendon fibrillae occupying
the intervals between adjacent extremities assume their position
at a stage in the developmental cycle later than the appearance of
the definitive form of the muscle fibrillae. In other words, the
muscle fibrillae are already attached to the internal surface of the
sarcolemma of the intervals before the supplementary tendon fibrillae
grow into these intervals and become attached to the external sur-
face of the sarcolemma. Accordingly, we can find another argument
in this fact of genesis, in addition to the one based upon morpho-
logical grounds and mentioned before, against the acceptance of the
view that the tendon fibrillae effect continuity with the muscle
fibrillae by perforating the sarcolemma and then coursing a con-
siderable distance in the muscle fibre. Indeed, were structural con-
tinuity an established fact, if the chronological order of the develop-
ment of these »interval« tendon fibrillae and of the muscle fibrillae
were alone considered we should expect rather to find a prolonga-
tion of a portion of undifferentiated muscle fibrillae through and
outside of the sarcolemma in order to meet the developing tendon
fibrillae, and not vice-versa as some authors have represented. The
presence of extrasarcolemmatous muscle fibrillae has never been ob-
served among vertebrates, so far as I am aware, and in my own
preparations is most positively denied by the staining reactions.
The only fibrillae lying outside of the sarcolemma are those which
have a connective tissue origin.

In general it may be said, then, that the voluntary striped
muscles of adult vertebrates terminate in one of two general
arrangements the determination of which is dependent upon tlic
relation of the long axis of the tendon to that of the muscle fibre.

258 W. M. Baldwin

When the direction of the two coincide, the muscle fibre of the

adult retains its earlier developmental features to the extent that.

the sarcolemma end still preserves its cone-shaped blunt projections
into which the muscle fibrillae, presenting all of their features of
cross-striation, enter to fuse with its internal surface. These sarco-
lemma projections with their intervening recesses are dovetailed into
corresponding features of the tendon extremity. The second general
type of muscle end is observable in those other muscles where the
long axis of the tendon meets that of the muscle fibre at an angle.
This form is to be regarded as a developmental derivative of a con-
dition which in the younger specimens conformed to the type of
structure which we have designated under I. So far as my own ob-
servations have led me I have not yet seen a developing muscle
fibre which did not conform to the first type.

In other words, before the muscle fibre has grown far enough
to reach its definitive oblique insertion it terminates in cone-shaped
sarcolemma processes which give attachment to tendon fibrillae whose
direction corresponds to the long axis of the muscle fibre itself.

Those muscles conforming to the bipenniform type of arrange-
ment present the most positive evidence against a continuity of
muscle fibrillae with tendon fibrillae, since in these a layer of dense
connective tissue, the peritendinum, intervenes and separates the two
structures from each other. The features to be considered with
especial care in these muscles, therefore, are the peritendinum
fibrillae and the muscle fibrillae. Continuity between the latter and
the tendon fibrillae is absolutely out of the question. The presence
of the greatly thickened sarcolemma end whose homogeneous nature
can be very readily made out with a magnification of 1500 dia-
meters, renders possible the most definite answer to the question
of continuity so far as these muscles are concerned. In no instance
is there the slightest indication of a fibrillar structure perforating
this homogeneous membrane. There is surely no turning of the
peritendinum fibrillae observable as must be the case were conti-
nuity to be established with the obliquely lying muscle fibrillae.
We are in a position, therefore, so far as these muscles of the
adult are considered, to corroborate most completely the views of
RANVIER, WEISMANN, K6LLIKER and Morro-Coca, that no conti-
nuity exists between the tendon fibrillae and the muscle fibrillae.

The same conclusions may be drawn as well in the instance of
those other muscles conforming to this general type where there

--

The Relation of Muscle Fibrillae to Tendon Fibrillae ete. 959

are no peritendinum fibrillae intervening between the tendon and the
obliquely-placed muscle fibres. Explaining upon developmental
grounds the presence of the sarcolemma infoldings into the muscle
fibre, and ascertaining through staining reactions the identity of
these infoldings in the adult with the sarcolemma, and, in addition,
by the same methods most positively establishing the morphological
differences between such structures and the muscle and tendon
fibrillae, and moreover, failing to observe any turning of the tendon
fibrillae at an obtuse angle, as would be necessary to establish con-
tinuity with the muscle fibrillae, we are in a position here, as well,
to deny in this type of muscle the continuity of tendon fibrillae with
muscle fibrillae.

Regarding those other muscles which conform to the first type
of termination, where the linear direction of the tendon fibrillae in
the adult corresponds to that of the muscle fibrillae, it must be con-
fessed that the problem is not so readily solved, the answer, ho-
wever, can just as positively be given. The chief difficulty encoun-
tered is the exceeding thinness of the sarcolemma and the overlying
and underlying of tendon and of muscle fibrillae in vertical optical
planes upon the obliquely inclined surfaces of sarcolemma-end pro-
cesses. The two kinds of fibrillae accordingly lie closer together.

The facts pointing strongly against continuity in this type of
muscle are, — each sarcolemma process has attached to it but a single
connective-tissue fibril, yet from ten to forty muscle fibrillae termi-
nate in the respective process. A reduction in diameter of each
muscle fibril is not demonstrable, such as must be the case if conti-
nuity with the single attached tendon fibril existed. Furthermore,
there is no indication of a change in direction of the muscle fibrillae
at the sarcolemma end in order to join the single apically-attached
tendon fibril. The sarcolemma of the process is not thickened as
one might expect to find, if the numerous muscle fibrillae turned
and passed along its surface or through its substance in order to
reach the tendon fibril. Again, the differences in size of the tendon
fibrillae are not correlative to the variations in size of the sarcolemma
processes or to the number of muscle fibrillae therein terminating.
Further, the single tendon fibrillae can be traced up to a connective
tissue cell body, a fact suggestive of their genesis. The staining
reactions prove that such fibrillae pass only up to the sarcolemma
end. In addition, in adult muscles the cross-striation proceeds di-
rectly up to the internal surface of the cone-shaped sarcolemma

260 W. M. Baldwin

processes. ‘This fact alone speaks most positively against a perfo-
ration of the sarcolemma by the tendon fibrillae and their subsequent .
passage through the muscle fibre to meet and fuse with the muscle
fibrillae. Moreover, there are at first no tendon fibrillae attached to
the sarcolemma end in the intervals between the cone-like processes,
whereas the muscle fibrillae, undifferentiated in structure are almost
from the very first already attached to the internal surface of the
sarcolemma at such corresponding intervals.

‘A word should be said about the combination of fuchsin 8 with
aleoholic-hematoxylin as recommended by several investigators. I
have used these stains upon many of my own preparations, with the
result that, dependent upon the relative concentration of the former
constituent and the time of exposure, I was able to stain with the
fuchsin not only the tendon fibrillae but also that portion of the
terminal undifferentiated muscle fibrillae lying adjacent to and atta-
ched to the sarcolemma end. Indeed, by carrying the staining a
little further I was able to stain the neighboring portions of the
muscle fibrillae which presented all of the features of cross-striation.
Hence, this combination of stains seems to be most unreliable as a
criterion of morphological values.

For the developmental and morphological reasons which I have
enumerated above, the assertion, that these ends of undifferentiated
muscle fibrillae, by reason of their staining reactions, represent ten-
don fibrillae which have perforated the sarcolemma in order to join
the muscle fibrillae, cannot be accepted as convincing to say nothing
at all about being most positively denied. Yet some of the publi-
shed figures intended to represent the continuity of these two kinds
of fibrillae and stained with the same stains give exactly this same
appearance.

In order to be positive that the cone-shaped extremities of the
sarcolemma end were not the result of shrinkage of the tissue in
the course of its preparation, I studied specimens of living muscle
under high magnifications and ascertained that these processes were
characteristic of the termination of the muscles conforming to the
first type of ending. I found also that the presence of a uni-
formly rounded sarcolemma end such as has been figured by other
investigators may be accepted as proof positive that the actual end
of the muscle fibre has not been represented. I shall have occasion
to refer to this feature in connection with fig. 11 upon a subsequent

page.

The Relation of Muscle Fibrillae to Tendon Fibrillae ete. 961

I have presented fig. 10 for the purpose of elucidating certain
appearances which have been misinterpreted by various inyesti-
gators who have endeavored to establish a continuity between muscle
and tendon fibrillae. The figure bears, as can be readily noted, a
close resemblance to some of their published plates. This figure
was sketched from a specimen of thigh muscle of an adult white
mouse. Both the tendon fibrillae and the muscle fibrillae pursued
the same linear direction. Upon the left side of the fibre the sarco-
lemma surface (6) at the end of the muscle fibre lay in a plane at
exactly a right angle to that of the fibrillae, while on the right
side it encountered these structures at an acute angle. These two
portions of sarcolemma are uninterruptedly continuous with each
other (B—B). The left side of the fibre demonstrates all of those
features to which I have previously referred. The tendon fibrillae
are separated from the muscle fibrillae by the thickened sarcolemma
end. Upon the right side of the fibre the outline of the sarco-
lemma because of its obliquity is with much greater difficulty ob-
served. Those fibrillae which occupy the same optical plane, for
instance, the uppermost aspect of the section, can be seen to be
separated from each other by the thin cut edge of the sarcolemma.
Those other tendon fibrillae which occupy the middle of the thick-
ness of the specimen seem to have perforated that membrane and
to have extended into the muscle fibre. This appearance is natur-
ally referable to the fact that they are attached to the under sur-
face of obliquely inclined sarcolemma and hence underlie the upper-
most muscle fibrillae. Their intense red stain, derived from the
fuchsin, lends its color, too, to these adjacent overlying muscle
fibrillae and, consequently, heightens the impression that a portion
of them extends into the muscle fibre. A careful consideration of
the left side of the sketch is sufficient, however, to eradicate all
doubt of the discontinuity of the two kinds of fibrillae.

The particular criticism that I would raise against O. SCHULTZE’s
work in that he has neglected to explain an appearance which is
represented in almost every one of his figures, and which is of
fundamental importance in our conception of the relation of the
sarcoplasm to the sarcolemma. At places he has represented groups
of three, four, and more muscle fibrillae which together perforate
the sarcolemma and which are then prolonged as tendon fibrillae,
i. e., the sarcolemma is interrupted at the point of perforation of
not single muscle fibrillae but of groups of fibrillae. In other words,

262 W. M. Baldwin

the intervening sarcoplasm, as well as the fibrillae, is continued
beyond the limits of the sarcolemma end outside of the muscle fibre. —
Still no attempt is made to explain with what the sarcoplasm be-
comes contiuous or where it ends. Were the condition true, as
the author has figured, then we should be compelled to modify our
conception of the sarcolemma as a closed tube or envelope confining
the semifluid sarcoplasm.

I have studied my own tadpole preparations with this parti-
cular point in mind and have represented in fig. 11 a muscle termi-
nation which may be considered as typical of the developing fibres
at this age. The tadpole measured 1,5 cm.

I agree for the present with other investigators in naming the
nucleus, which is seen imbedded in the granular protoplasmic mass
surrounding the muscle fibrillae, a myogenetic nucleus, and also in
referring to the investing membrane as sarcolemma (B). I have
carried this membrane around the muscle fibre end, since such is the
appearance produced when focusing down upon the fibre. It does
not represent the end of the muscle fibrillae, however, as might at
first appear, even in spite of their undifferentiated appearance. This
fact can. be ascertained by focusing to a deeper level in the fibre,
then we get the appearance such as I have represented. The muscle
fibrillae terminate in a number of cone-shaped processes, whose
walls are formed of a delicate membrane, continuous with the sarco-
lemma, which bridges the ends of those fibrillae. This appearance
is similar of that observed in the muscle fibre represented in fig. 6. These
are the processes and this the membrane which have been over-
looked. The specific remarks which I have made upon foregoing
pages regarding the features to be noted in connection with fig. 6
apply equally well here and require no repetition.

The presence of this unbroken rounded contour of sarcolemma
traversing the end of the muscle fibre, to which I have referred
above, is readily explained, if we will imagine the muscle fibre as
a whole rotated through an are of 90°. Where the sarcolemma
surface faces the eye it is almost perfectly transparent, but where,
however, it lies in a vertical optical plane, its contour becomes
manifest. Hence, the rounded sarcolemma-end appearance would be
demonstrated again in the rotated condition of the fibre by that
portion located at A in the figure. At the same time we ean rea-
dily understand how the muscle fibrillae lying at a deeper level
appear to have passed outside of the sarcolemma. And when through

The Relation of Muscle Fibrillae to Tendon Fibrillae ete. 263

imperfect fixation or staining we overlook the exceedingly delicate,
definitive, cone-shaped, sarcolemma processes, the conclusion is natural
that the tendon fibrillae and muscle fibrillae are continuous because
the sarcolemma has been perforated. This figure answers the
question, as well, as to the relation of the sarcoplasm to the sarco-
lemma. Our earlier conception of the latter as a closed tube
enclosing the sarcoplasm is correct. At no place does the sarco-
plasm pass through the sarcolemma. At every point this thin enve-
lope separates the specialized, semifluid sarcoplasm from the inter-
stitial fluids, lymph, ete., of the outlying tissues.

I have not been able to carry out my studies on either Hippo-
campus or Amphioxus. It would be interesting to explain upon
phylogenetic and ontogenetic grounds the differences in the adult
morphological condition in these vertebrates from that which I have
described above in higher vertebrate muscles.

As a final word upon the question of the cone-shaped processes
of sarcolemma I desire to repeat with added emphasis, lest some
criticisms regarding faulty fixation or shrinkage be made, that as
controls to the fixed and stained preparations I studied many prepa-
rations of living muscle by vital staining in monochromatic light.
Methylene blue aqueous solutions among others were used and of
such a degree of concentration that a control tadpole lived for
thirty-six hours in the same fluid which was used to stain the cau-
dal muscles. The preparations which I employed were studied ten
minutes after the removal of the muscle. No fixatives or dehydra-
ting agents whatever were used. In every one of these living
specimens the cone-shaped sarcolemma processes could be readily
found. Therefore, they did not owe their presence to an arte-
factitious change in the muscle structure.

The following general conclusions, then, regarding these various
voluntary striped muscles of the tadpole, white mouse, gray mouse,
chicken, frog, and calf may be drawn.

1st — In the manner of termination of muscle fibres two general
types may be recognized, one in which the long axes of the tendon
and of the muscle fibres coincide, and the second in which they
meet at an angle.

2nd — In neither of these two types are the muscle fibrillae
in continuity with the tendon fibrillae.

3rd — Developing muscle fibres terminate in a number of cone-

264 W. M. Baldwin

shaped processes of sarcolemma to the apex of which a tendon fibril
is attached.

4th — In the adult those muscles conforming to type 1 still pre-
serve the apical processes of their sarcolemma end.

5th — In the adult these processes of sarcolemma are dove-
tailed into the tendon end.

6th — ‘The sarcolemma at the tendon end of such muscles is

not markedly thickened.

’ 7th — The central tendon of bipenniform muscles (type 2) is in-
vested by a connective tissue sheath or peritendinum which consists
of connective tissue fibres and cells, and which separates the tendon
fibrillae from the muscle fibres.

8th — On muscles, conforming to the second type of structure,
the sarcolemma end presents a flat surface which rests directly
against the attached structure, be it peritendinum, perichondrium, or
periosteum.

9th — This sarcolemma end is considerably thickened and is
composed of a homogeneous substance.

10th — It presents a number of sarcolemma projections which
project into the substance of the muscle fibre.
11th — These projections are derived from the fused, adjacent

walls of the cone-shaped processes of the sarcolemma which were
present at an earlier developmental stage of the fibre.

12th — The muscle fibrillae in adult muscles of this second
type preserve their features of cross-striation up to the sarcolemma.

13th — This sarcolemma end is not perforated either by the
tendon fibrillae, the peritendinum fibrillae, or the muscle fibrillae.

14th — The sarcolemma is not prolonged through the tendon,
or over the tendon in either type of muscle.

15th — The sarcoplasm does not at any place in either type of
muscle pass through the sarcolemma.

Literature.

GAGE, S. H., The Microscope. 10th Edit. The Comstock Publ. Co., Ithaca,
N- ¥.2 1908:

KOLLikER, A., Handbuch der Gewebelehre des Menschen. Leipzig 1889.

Morra-Coca, A. and Frruiro, C., Contributo allo studio dei rapporti tra mus-
coli e tendini. Monitore zoologico. Vol. X. 1899.

The Relation of Muscle Fibrillae to Tendon Fibrillae ete. 265

Ranvier, L., Traité technique d’Histologie. 2nd Edit. 1889.

ScHIEFFERDECKER, P., Untersuchungen iiberdie Rumpfmuskulatur von Petromyzon
fluviatilis, ete. Archiv f. mikrosk. Anat. Bd. LXXVIII. iB alale

Scuuttze, O., Uber den direkten Zusammenhang von Muskelfibrillen und Sehnen-
fibrillen. Arch. f. mikrosk. Anat. Bd. LXXIX. 1912.

Weisman, A., Uber die Verbindung der Muskelfasern mit ihren Ansatzpunkten.
Zeitschr. f. rationelle Medizin. Bd. XII. 1861. 5

Explanation of figures.

Plate VII.

Fig. 1. An isolated muscle fibre with its attached tendon of an extrinsic
muscle of the eyeball of a three-weeks-old chicken. The dovetailing
of the sarcolemma end with the tendon fibrillae is characteristic of
muscles belonging to this general type. The muscle fibrillae do not
present the features of cross-striation at the muscle end. Magnification
1000 diameters.

Fig. 2. Similar to fig. 1 excepting that the muscle fibril groups are separated
from each other by two nuclei. Magnification 1000 diameters.

Fig. 3. A muscle fibre from an extrinsic eye-muscle of a white mouse twenty-
two days old. It presents the same general features as the two pre-
ceeding figures. Four tendon fibrillae (A), are to be noted upon the
uppermost aspect of the sarcolemma end. Magnification 1000 diameters.

Fig.4. A portion of a muscle fibre and tendon of a thigh muscle of an adult
white mouse, which demonstrates the kind of muscle ending com-
prised under group 2. The tendon fibrillae (A), are separated from
the muscle fibrillae (0), by the peritendinum (8), which contains four
cells. Magnification 1000 diameters.

Fig. 5. Similar to the preceeding. The features of cross-striation can be
traced up to the thickened sarcolemma where each is inserted upon
a small raised elevation (5) of the latter. Magnification 1500 diameters.

Fig. 6. A portion of a muscle fibre of a caudal muscle of a tadpole about
5,0em long. Each cone-shaped sarcolemma process has attached to
it a tendon fibril. Two of the processes derive fibrillae from a large
fibroblastic cell situated among the tendon fibrillae. The original
preparation from which this sketch was made demonstrates no thickening
of the sarcolemma forming these processes.

Fig. 7. A developing intercostal muscle ofa five-day old white mouse present-
ing three cone-shaped sarcolemma processes, each affording attachment
to a connective tissue fibril. The fibril on the right is observed to
be directly derived from a cell-body. Magnification 1000 diameters.

Fig. 8. Another developing fibre from the same muscle as the preceeding. In
this fibre a more advanced stage of development is seen. Part of
the muscle fibre is already attached to the perichondrium of the
developing rib-cartilage. Three sarcolemma processes upon the right
side of the fibre, however, have not yet reached their definitive attach-
ment, but present connective tissue fibrillae affixed to their apex.
Magnification 1000 diameters.

Moerpholog. Jahrbuch. 45. 17

266 W.M. Baldwin, The Relation ot Muscle Fibrillae to Tendon Fibrillae ete.

Fig. 10.

Fig. 11.

A semi-diagrammatic sketch of a developing intercostal muscle illu-

strating the pointed processes of sarcolemma each with its attached ©

tendon fibril. The features of cross-striation of the muscle fibre are
wanting at the sarcolemma end. Magnification 1000 diameters.

A thigh muscle fibre and tendon of an adult white mouse conforming
to type 2. Upon the left side of the figure several inturned processes
of sarcolemma are represented in the muscle fibre (A). Upon this
side the sarcolemma is cut exactly transversely. Upon the right side
of the figure the sarcolemma (PB) is encountered obliquely by the
section knife, consequently the more superficial of the muscle fibrillae
are underlaid by the tendon fibrillae. The sarcolemma separates the
two structures, still it appears as if the tendon fibrillae extended
among the muscle fibrillae having perforated the sarcolemma. Magni-
fication 1000 diameters.

This is a portion of a muscle fibre from a caudal muscle of a tad-
pole 1,5 cm long. The presence of the richly granular protoplasmic
mass imbedding a nucleus and surrounding the muscle fibrillae may
be taken as an indication that the muscle fibre is in a developmental
stage. In the adult state such protoplasmic masses are wanting.
The membrane (B) investing the mass may be provisionally inter-
preted as the sarcolemma. The presence of the line continuous with
the sarcolemma and carried across the fibre end does not represent
the fibre end. I have elucidated this appearance upon a previous
page (4). The sarcolemma proper is drawn out into a number of
processes in which the muscle fibrillae end and upon whose apex a
tendon fibril is inserted. Magnification 1500 diameters.

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Amtliches Organ der Anatomischen Gesellschaft.
Herausgegeben von Prof. Dr. Karl yon Bardeleben in Jena.
Verlag von Gustav Fischer in Jena.

42. Band, Nr. 7/8, 1912.

Nachdruck verboten.
Muscle Fibres and Muscle Cells of the adult White Mouse Heart.
W. M. Batpwiy.
(Cornell Medical College New York City.)
(From the Biological Laboratory at Bonn.)
With 2 Figures.

In two recent publications‘) the view was advanced as the result
of studies upon various voluntary striped muscles, such as latissimus
dorsi, rectus abdominalis, thigh and leg muscles and extrinsic eye
muscles of the chicken, cat, calf, white mouse, gray mouse, frog, and
caudal muscles of the tadpole, that these muscle fibres of the adult
should not be considered, as we have generally hitherto supposed, as
large multinucleated cells, but rather as composite contractile struc-
tures composed of muscle fibrillae and sarcoplasm and containing
muscle cells. That is to say, each one of the numerous nuclei which
seem to be immediately imbedded in the sarcoplasm, represents in
reality a distinct muscle cell, which presents cellular protoplasm com-
posed of a spongioplasm network with interstices of hyaloplasm and
which is completely invested by a cell membrane. This cell mem-
brane intervenes between the protoplasm of the cell, containing the
nucleus, and the imbedding sarcoplasm of the muscle fibre. In other
words, since these same relations hold which all muscle cells, the
muscle fibrillae and sarcoplasm are extra-cellular structures. Hence
the generalization usually held, that the adult voluntary striped muscle -
fibre is a multinucleated cell, as stated before, is erroneous.

Still another fact bespeaking the correctness of these assertions
was adduced from the studies of the sarcolemma in similar muscles
and detailed in the second communication. It was found in this
series that the sarcolemma was a structureless cuticula containing
neither cells nor fibrils. Kyerywhere it stood in direct contact with
the sarcoplasm of the fibre and afforded attachment to the telophrag-
mata. Furthermore, its relation to the peripheral-lying muscle cells
was such, that it was indented into the fibre by the latter, lying,
therefore, between them and the sarcoplasm and not upon the peri-

1) Zeitschrift fiir Aligem. Physiologie (Max Verworn) 1912.

178

pheral fibre-aspect of these cells. The one structure found to occupy
this position was the cellular and fibrillar investing perimysium of
the muscle fibre. Hence such muscle cells lie outside of the sarco-
lemma. The latter envelope encloses only the highly specialized
muscle fibrillae with the semi-fluid sacroplasm. Therefore, for these
additional reasons the muscle fibrillae are to be regarded in the
adult as extra- or intercellular structures.

An analogy between the histogenetic cycle of the connective
tissue group and that of voluntary striped muscle can be drawn.
The extremes of the cycle of the latter have been established by a
number of competent observers. The intermediate steps, however,
require further study. At first the myofibrillae are laid down in the
genetic cell bodies. At the opposite end of the genetic course these
fibrillae are extracellular. Parallel is the course of development in
the connective tissue group; at first appearing as intracellular fibrillae
and later being extruded from these genetic cell bodies. These facts
obtain as well in the instance of cardiac musculature of the adult
white mouse.

In the study of this form of striped muscle fibre the same
technic was employed as that detailed in the cited papers. The sec-
tions varied in thickness from 2 py. to 31/, p.; the stain used was
alcoholic hematoxylin.

The first figure represents a longitudinal section of several
muscle fibres of the ventricle. The marked morphological structural
differences between the protoplasma immediately investing the nuclei
and the sarcoplasm of the fibre are apparent at first observation. The
cellular spongio-plasmatic network of the former is wanting in the
latter. These two forms of protoplasm do not blend with each other;
rather, they are sharply delimited from each other by a distinct mem-
brane, the cell wall. With the alcoholic hematoxylin, this structure
stained deeply in contrast to the neighbouring parallel-running muscle
fibrillae. In focusing down through the section the uninterrupted,
membrane-like nature of the cell wall could be readily noted. In
contrast to this fact the slender muscle fibrillae pass into and out of
focus as their level was reached and passed. Again, the features of
cross-striation were not observed on the cell wall, hence the identity
of the membrane apart from the muscle fibrillae was established.

The question of the longitudinal extent of such cells, as was the
case with similar sections of voluntary striped muscle, is still un-

ne

answered. At the best the solution is exceedingly difficult. The
delicacy of the cell wall, the overlying or underlying of it by muscle
fibrillae, granulae, and telophragmata, all add to the difficulty in
answering the question. The middle of the three muscle cells in
fig. 1, however, possibly presents evidence upon which a correct
conclusion may be drawn. The uppermost pole of this cell demon-
strates what appears to be the reflected edge of the cell wall. The
instance is not exceptional, since many such appearances are demon-
strable throughout the entire series of sections. But the possibility
of its being an obliquely-sectioned cell extremity together with the
difficulties enumerated above render, with
our present microscopical technic, a posi-
tive answer most injudicious.

Two muscle fibres, represented as
transversely sectioned, are seen in figure 2.
A blood vessel occupies the angle between
them. Each fibre presents a muscle cell.
That on the left was encountered at the
level of the middle of the nucleus; that
on the right above the level of that
structure. In the latter the structure of
the cell protoplasm is in marked contrast
to that of the sarcoplasm. The presence
of a cell wall separating the two is un-
questionable. The spongioplasm network
of the cell is relatively heavily laden
with granules. Notwithstanding, the clear Fig. 1.
fibrillae of this network can in many
levels be traced directly up to the internal surface of this cell wall
upon which they end. They do not at any place find an insertion upon
the muscle fibrillae. A narrow interval of sarcoplasm, equal in general
to the cross-diameter of an average muscle fibril, intervenes between
the latter and the cell wall.

The cell on the left is of interest chiefly because it demonstrates
appearances comparable to those observed in connection with similarly
cut sections of voluntary striped muscles. The remarks made in the
cited articles regarding such appearances apply here as well. At
such levels the membrane appears to be wanting, i. e., the nucleus
seems to be immediately imbedded in the sarcoplasm. Hence the

Peele 8 SES Se Nalicd ny CSL BALM yO

~

ee

180

view generally held that the muscle fibrillae are intracellular, being
contained in a giant, multinucleated cell. Sections. above or below
the level of the nucleus, however, demonstrate the cell wall comple-
tely circumscribing the cell protoplasm. The inference seems to be
justifiable, furthermore, that it is not wanting at the level of the nucleus.
In torn preparations where the nucleus has been mechanically remoy-
ed from its’ intimate position in relation to the sarcoplasm, and in
those other preparations where the nucleus is shrunken, the presence
of a distinct wall continuous with the remaining portions of the cell
wall can be detected. It appears not to be an artefactitious product, since
its outline is regular and definite and it is uniformly and deeply stained.
Were it due to retracted and shrunken protoplasm we should expect

to find it irregular in outline, varying in

MGS SEES thickness, and differing in different levels

[SQ SS Sfx", in intensity of staining. It presents none
y ' of these artefactitious criterions. Nor does
it in any portion of its extent seem to be
merely the free, unthickened edge of the
sarcoplasm. It possesses on the contrary
a definite contour and a definite staining
reaction. Owing merely to the juxtaposition of the nucleus its out-
line is overlooked in normal unshrunken tissues. Naturally, therefore,
at such levels no spongioplasm fibrillae are attached to its internal
surface.

The two sketches in figure 1 and 2 are not intended to represent
exceptional instances in cardiac muscle structure. Such relations
were observed throughout the entire series of cardiac musculature
where the conditions were favorable for sharp observation, i. e., where
the parts concerned were not obscured by the overlying or under-
lying by granulae, muscle fibrillae, telophragmata, &c.

In the study of the telophragmata of this type of muscle no
instance was observed where these lines traversed either the proto-
plasm or the nucleus of the muscle cells. This fact may be inter-
preted as of the following significance. First, it bespeaks the con-
tinuity of the cell protoplasm and nucleus as appertaining to a distinct
and individual morphological and functional unit, a cell. Further-
more, granting the correctness of the observations of numerous
workers that the telophragmata are always directly and uninterruptedly
inserted upon the interna] surface of the sarcolemma, we should look

2

Fig. 2.

1814
for an infolding of the sarcolemma from the periphery of the muscle
fibre to invest these cells. No definite proof has been ascertained
as yet that such is the instance in the cardiac fibres. The presence
of an investment of sarcolemma upon the muscle cells is not, however,
negatived by this fact. The telophragmata are attached, apparently,
directly to the external surface of the cell membrane. This fact
cannot be adduced as conclusive evidence, notwithstanding, arguing
against the verified observations mentioned above. The matter
demands further observation upon a greater number of vertebrates.

Much remains to be studied upon the intermediate genetic steps
of histo-myogenesis. Sufficient evidence of the presence of myo-
fibrillae as intra-cellular structures in the first developmental stages
exists in the literature. In the adult stages of both forms of striped
muscle, so far as concerns these particular vertebrates investigated,
these muscle fibrillae are extracellular. A parallelism can be drawn,
therefore, between myogenesis and developmental sequence observed
and, seemingly, well established in the case of the connective tissue
group of structures. At first the connective tissue and the elastic
tissue fibrillae are intracellular. Later in development they are
extruded from the genetic cell bodies and occupy, an intercellular
position. Such is the sequence as well with the striped muscle
fibrillae. In this fact we can find an additional reason, first, for
grouping these striped muscle fibres, voluntary and cardiac, among
the connective tissue group of structures, and secondly, for not con-
sidering them as multi- or singly-nucleated giant cells. In other
words the muscle fibrillae and sarcoplasm are inter- or extracellular
structures. To this extent these observations corroborate those made
upon the voluntary muscles and detailed in the articles cited.

The conclusion arrived at, then, is that our conception of the
cardiac muscle fibre as a cell containing fibrillae and sarcoplasm is
erroneous as far as concerns the adult white mouse. The terms muscle
fibre and muscle cell are not synonymous. The cuticular sarcolemma
invests both the highly specialized muscle fibrillae and the sarcoplasm
and, in addition, muscle cells. The latter structures present a nucleus,
cell protoplasm, consisting of a spongioplasmatic network with inter-
stices of hyaloplasm, and a cell wall. By reason of the last the

cells are everywhere excluded from the sarcoplasm and the muscle
fibrillae.

bo
|
re

Aus dem biologischen Laboratorium der Universitat Bonn.

Die Entwicklung der Fasern der Zonula Zinnii im

Auge der weissen Maus nach der Geburt.
Von
W. M. Baldwin

Instructor in Anatomy. Cornell University Medical College. New York City.

Hierzu Tafel XIV und XY.

Der Gegenstand dieser Untersuchung war die Bestimmung
des Ursprungs, der Entwicklung und der endgiiltigen Anordnung
der Zonula Zinnii- Fasern des Saugetierauges, indem ich fiir
diesen Zweck eine doppelte Reihe von weissen Mausen benutzte,
im Alter von 12 Stunden bis einschliesslich 17 Tagen, mit anderen
im Alter von 22, 27 Tagen und ausgewachsenen Exemplaren.
Dazu hatte ich noch Gelegenheit, Exemplare der Katze. des
Kalbes und des ausgewachsenen Menschen zu studieren. Die
meisten meiner Resultate aber griinden sich auf meine Studien
an weissen Mausen.

Die verwendeten Fixiermittel waren die Flemmingsche
Loésung und Sublimat. Die Einbettung geschah nach der Paraffin-
methode. Alle Schnitte wurden in einer Richtung gemacht, aus-
strahlend vom Mittelpunkte der Linse, und in einer Dicke von
2,9—7,9 uw. Die von mir benutzten Farbemittel waren Safranin,
Orcein, Chloralhimatoxylin (Gage) und Bielschowskys Nerven-
faserfarbung.

Der erste Teil meiner Arbeit behandelt die Erscheinungen
bei 12 Stunden, 5, 11 und 14 Tage alten Exemplaren; im zweiten
Teile soll ein fortschreitendes Bild der in der Entwicklung ent-
stehenden Veranderungen gegeben werden, welche die besonderen
Gebilde, die in den Bereich unseres Problems fallen, durchzumachen
haben, bis sie zu ihrer bleibenden Gestalt und Lage gelangen:
im dritten Teile endlich werde ich kurz die Ansichten anderer
Forscher, die Entstehung der Fasern betreffend, anfiihren und
nach meinen eigenen Beobachtungen die Griinde fiir und gegen
die Aufrechterhaltung dieser Ansichten ausfiihrlich erértern.

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Die Entwicklung der Fasern der Zonula Zinnii. 27

Die weisse Maus, 12 Stunden alt.

Die Augenlider sind noch nicht gedffnet. Die Netzhaut ist
in ihrer Entwicklung so weit vorgeschritten, dass mebrere be-
stimmte Schichten identifiziert werden kénnen; aber die Schicht
der Zapfen und Stabchen. ist noch nicht vorhanden. Der Glas-
kérperraum ist von einer Menge von Fasern derart durchzogen,
dass sie ein Netzwerk bilden, welches viele Zellkérper und Blut-
gefasse enthalt. Dieses Netzwerk reicht, wie man sehen kann,
distalwarts') bis zur inneren Oberflache der Linse und dringt
seitwirts von ihr in den Raum zwischen der pars ciliaris retinae
und der Linse ein.

Uberall, obgleich in der letzten Region weniger bemerkbar,
ist auf diesem Netzwerk ein kérniger Niederschlag zu beobachten,
der sich stark mit Himatoxylin farbt. Dieser Zustand verdunkelt
den feinen Bau der Fasern, der Zellen und der begleitenden
Blutgefasse. Die Zellen sind gross, hell und spindel- oder stern-
formig und enthalten einen verhaltnismassig kleinen Kern. Ihre
Ausliufer bilden das kérnige Netzwerk. Mehrere solche Zellen
sind auf der inneren Oberflache der limitans retinae interna zu
erkennen, lings welcher ihre Fasern zusammen mit anderen, vom
Netzwerk ausgehenden laufen. Diese Fasern aber verbinden sich
nicht mit der limitans. Die Blutgefiisse, in diesem Alter ver-
haltnismassig sehr zahlreich, liegen auf der limitans, oder sie
durchziehen das Netzwerk, werden von ihm getragen und erreichen
die Linse, auf der sie ein besonders reiches Netz bilden.

Die aussere Oberfliche der limitans stellt eine scharfe Linie
gegen das helle Protoplasma der darunter liegenden Netzhaut-
zellen dar, in starkem Gegensatz zu ihrer inneren Oberfliche
mit den dazukommenden, aus dem Glaskérpernetzwerk stammenden
Fasern. Die limitans selbst erscheint als eine deutliche, dicke
und sich dunkel farbende Membran. Bei einigen Schnitten traf
ich gliicklicherweise ihre flache Oberfliche, so dass ihr Bau gut
zu untersuchen war. Es fehlten ihr sowohl Zellumrisse als auch
Fasern. Diejenigen Fasern des Glaskérpernetzwerkes, welche sich
langs ihrer inneren Oberfliche hinziehen, schienen an keiner

*) In dieser ganzen Arbeit habe ich die Ausdriicke distal“ und
,proximal* angewendet; ersterer bedeutet an dem, oder in der Richtung auf
den Hornhautpol des Augapfels hin, letzterer an oder in der Richtung zum
Netzhaut- oder Funduspol hin.

976 W. M. Baldwin:

Stelle mit ihr zu verschmelzen oder in ihr inneres Gefiige ein-
zudringen. Sie ist durchweg vollstandig homogen. Verfolgt man
sie distalwarts, so findet man, dass sie auf der Hohe der ora
serrata in mehrere Schichten und Fasern sich auflést, yon denen
jede sich bis zur Spitze einer ciliaren. Epithelzelle der inneren
Schicht fortsetzt. Die Linsenhéhle besteht noch; aber die Zellen
der durchsichtigen oder proximalen Reihe sind schon in lange
Siulen ausgezogen. Die Kapsel erscheint auf beiden Seiten der
Linse als eine verhaltnismiassig dicke fibrillare Membran, welche
ein reiches Netzwerk von anastomosierenden Blutgefiassen trigt.
Zu bemerken ist, dass die proximale Oberflache der Linse schon
konvexer als die distale ist. Die Hornhaut zeigt drei Schichten,
von denen die mittelste viele lingliche Zellen mit dunkel sich
firbenden Kernen enthalt. Die Regenbogenhaut hat sich soweit
entwickelt, dass sie nun neben ihrer zweischichtigen Epithellage
auf der proximalen Seite eine deutliche Schicht von Mesenchym-
gewebe zeigt, in welchem an dem pupillaren Rande der Iris die
Anlage des m. sphincter iridis zu erkennen ist. Die noch unver-
sehrte Pupillarmembran erstreckt sich durch den _ pupillaren
Zwischenraum, indem sie sich wohl an die distale Linsenkapsel
anlehnt, aber nicht mit ihr verschmilzt. Seitwiirts kann sie als
eine von einer einzelnen Endothelzellenlage gebildete Schicht
iiber die distale Seite des Mesenchymlagers der Iris bis zur
Verbindung dieser mit der Hornhaut verfolgt werden. Bei einigen
Schnitten ist in dem Raume zwischen dem Irisrande und der
Linse ein Blutgefiiss zu finden, welches sich bis zur distalen
Oberfliche der Linse hinzieht.

Die pars optica retinae setzt sich gegen die pars ciliaris
retinae an der ora serrata ab. Schon hat die Entwicklung der
Ciliarfalten begonnen, und man kann bemerken, dass sie einen
Kern yon Mesenchymgewebe einschliessen, welcher Blutkérperchen
enthaltende Gefisse besitzt. Das Ciliarepithel ist iiberall aus zwei
Schichten von Zylinderzellen zusammengesetzt. Hine wirkliche
limitans ciliaris interna dagegen fehlt. Auf der inneren Epithel-
oberfliche ist nichts zu sehen als der einfache, diinne Epithel-
rand dieser scharf gegeneinander abgesetzten Zellen. An dieser
Stelle muss noch besonders bemerkt werden, dass die Zellen der
inneren Schicht des Ciliarepithels in eine Spitze oder einen Fort-
satz auslaufen, und dass durch die ganze Linge der pars ciliaris

Die Entwicklung der Fasern der Zonula Zinnii. 277

retinae jede Zelle einen solchen spitzen Fortsatz besitzt. Solche
Fortsitze kénnen tatsichlich distalwarts bis zur inneren Seite
der Irisbasis bemerkt werden. Ferner ist zu erwahnen, dass alle
jene Epithelzellen zwischen der Spitze der definitiven Ciliarfalten
und der ora serrata eine Faser haben, die sich an ihre Spitze
ansetzt, dass aber in der Region distal von den erwahnten Teilen
viele spitze Zellen zu bemerken sind, welche keinen Faseransatz
haben. Von den distalen Verbindungen dieser Fasern und ihrer
Bedeutung werde ich spater ausfiihrlicher sprechen.

Die limitans retinae interna hort plotzlich an der ora ser-
rata auf. Sie ist nicht distalwarts tiber das Ciliarepithel verlangert.
Die limitans retinae externa ist indessen distalwarts zwischen den
beiden Schichten des Ciliarepithels zu verfolgen. Sie ist distal-
warts nicht so dick, erscheint jedoch trotzdem als eine deutliche,
gleichartige und ununterbrochene lamina.

Das Retziussche Biindel, das sich aus Fasern zusammensetzt,
die aus den Epithelzellen unmittelbar distal yon der ora serrata
hervorgehen und durch den ganzen Glaskérperraum strahlen, wie
leicht an Embryoschnitten zu erkennen ist, ist verschwunden
und an den Schnitten der 12 Stunden alten Maus nicht mehr
aufzufinden. Hier gibt es keine membrana hyaloidea, und dem-
gemass findet noch keine Trennung des Glaskérperraumes von
dem definitiven Zonularaum statt. Viele Blutgefiisse mit ihren
Blutkérperchen durchziehen diesen letzten Raum. Einige davon
setzen sich an die Linsenkapsel an, wahrend andere lings des
Ciliarepithels verlaufen. Die gréssten dieser Gefiisse aber sind
in der Mitte des Raumes zu bemerken, getragen von einer diinnen
ringformigen Membran. Diese reicht proximalwirts nicht tiber
die Ebene der ora serrata hinaus und kann distalwarts bis zu
jener Stelle der distalen Linsenoberfliche verfolgt werden, wo
die Gefasse, welche sie tragt, sich an die Linsenkapsel anlegen.
Bei einigen Schnitten liegt sie dicht an der Linse, bei anderen
in nachster Nahe des Ciliarepithels. Sie wird in diesen ver-
schiedenen Lagen von einer Reihe von Fasern gestiitzt, welche
an die benachbarten Teile sich anlegen.

Die Membran selbst ist diinn und farbt sich stark mit
Hamatoxylin. Bei starker Vergrésserung zeigt es sich, dass sie
aus einer oder zwei Schichten von Protoplasmafortsitzen gebildet
ist, die aus spindelférmigen oder vierseitigen Zellen stammen.

278 W. M. Baldwin:

Diese Zellen, welche in der ringférmigen, stiitzenden Membran
vorkommen, sich gelegentlich aber auch auf den Gefisswiinden
vortinden, enthalten einen runden oder ovalen Zellkern und
reichlich kérniges, sich dunkel farbendes Protoplasma. Gewéhnlich
gibt jede Zelle zwei Fortsitze ab, durch deren Vereinigung die
hier besprochene Membran gebildet wird. Gelegentlich ist indessen
auch nur ein Fortsatz einer spindelformigen Zelle zu bemerken,
welche zur Linse hiniitbergeht; aber diese Beobachtung ist nur da
zu machen, wo die Membran unterbrochen ist. Mitunter sieht
man auch einen ahnlichen Fortsatz, der zum Ciliarepithel iiber-
geht, wo er sich an die Spitze einer Epithelzelle anheftet. Solche
Fortsatze sind gewohnlich dick, mit Hamatoxylin dunkel gefiarbt
und besitzen einen kérnigen Niederschlag, abnlich wie er auf den
Fasern des Glaskérperraumes sich findet.

Noch ein anderer Zelltypus kann im Zonularaum_ nach-
gewiesen werden. Er ist jedoch anscheinend auf diese Region
beschrankt, da ich ahnliche Zellen im Glaskérperraum nicht finden
kann. Es sind dies grosse, unregelmiassige Zellen mit einem
grossen ovalen oder unregelmissigen Kern und reichlichem Proto-
plasma, welches sich fast gar nicht mit Hiimatoxylin farbt. Solche
Zellen liegen entweder auf der Gefaisshaut oder in dem leeren
Raum zwischen ihr und dem Ciliarepithel. Zwischen der Linse
und der Membran habe ich diese Zellen nicht gefunden. Jede
Zelle ist besonders gekennzeichnet durch die grosse Zah! von
Fortsitzen, die sie abgibt. Letztere sind ausserordentlich fein
und hell und farben sich nur leicht mit Hamatoxylin. Auch
ist auf ihnen kein kérniger Niederschlag zu bemerken.

Solche fadenartigen Fortsitze ziehen sich entweder lings
der stiitzenden, ringformigen Membran oder gegen das Ciliar-
epithel hin. Dagegen kann ich keine finden, die sich nach der
Linse hin erstreckten. Sie verzweigen und vereinigen sich oft,
wodurch sie ein dichtes und verworrenes Netzwerk von sehr feinen
Fasern bilden, welches zwischen der ora serrata und den Ciliar-
fortsitzen am dicksten ist. Aber nur wenige Faserchen dieses
Netzwerkes sind distal von dieser Gegend zu verfolgen. Jedoch
ist leicht zu beobachten, dass jedes Faserchen sich schliesslich
an die Spitze einer Ciliarepithelzelle festheftet. Solche, die in
den Zwischenriumen benachbarter Epithelzellen inserieren, kann
ich nicht finden, Die Fasern, welche von den spindelformigen

Die Entwicklung der Fasern der Zonula Zinnii. 279
Zellen der Gefassmembran entspringen, durchziehen dieses Netz-
werk, um an eine Epithelzelle zu inserieren, wie ich friiher
bemerkt habe; jedoch wegen ihrer Dicke, ihrer dunkeln-Farbung
und auch wegen des kornigen Niederschlages sind sie leicht von
den zarten, hellen, keine Korner fiihrenden Fasern des eigent-
lichen Netzwerkes zu unterscheiden. ;

Ein dritter Fasertypus endlich ist noch in dem Raume zu
erkennen. Dieser Typus ist indessen nicht oft zu bemerken,
sondern ist auf die Spitzen der Ciliarfortsitze beschrankt. Er
ist nur bei solchen Exemplaren zu beobachten, wo die Gefiss-
membran in nachster Nihe jener Gebilde liegt. Es ist ein kurzer
und verhiltnismiassig sehr breiter, schwach kérniger Protoplasma-
fortsatz, welcher von der Ciliarzellenschicht direkt zur Membran
geht, mit welcher er sich allem Anscheine nach vereinigt und so
einer weiteren Untersuchung sich entzieht. Die Fortsatze dieser
Art scheinen einfache Protoplasmafaden der Epithelzellen zu sein.

Die weisse Maus, 5 Tage alt.

Der Glaskérperraum zeigt in diesem Alter weniger Blut-
gefasse und Zellen, waihrend der kornige Niederschlag auf den
Fasern, welche den Raum durchziehen, so gut wie verschwunden
ist. Die verschiedenen Schichten der Netzhaut haben sich dem
Alter des Tieres entsprechend weiter entwickelt. Die lim. ret.
int. endet noch auf der Ebene der ora serrata, indem sie sich in
eine Anzahl von Schichten auflost, welche zu den Epithelzellen
gehen. Man kann iiberdies beobachten, dass jetzt mehr dieser
sie zusammensetzenden Schichten vorhanden sind als friiher, und
dass die Intercellularsubstanz zwischen den Epithelzellen der
inneren Ciliarschicht auch dicker ist. Indessen kann man keine
Abgrenzung dieser Intercellularsubstanz von dem gleichartigen
Bau der Schichten, welche die lim. bilden, erkennen. Die beiden
Gebilde stehen in direktem Zusammenhang miteinander. Bei
naherer Betrachtung scheinen die verschiedenen Lamellen sich
bei der Anniherung an die Fortsatze der Spitze der Epithelzellen
zu spalten, und jede Halfte einer geteilten Lamelle verbindet
sich dann sofort mit der benachbarten Intercellularsubstanz. Bei
noch weiterem Verfolgen nach der Seite findet man, dass diese
Intercellularsubstanz in direkte Verbindung mit der gleichartigen
Substanz der lim. ret. ext. tritt und damit verschmilzt. Zu der

280 W. M. Baldwin:

Feststellung ihres direkten Zusammenhanges miteinander kommt
hinzu, dass diese drei Gebilde: lim. ret. int., Intercellularsubstanz
der ora serrata und lim. ret. ext. auch wegen ihres Verhaltens
bei der Farbung und wegen ihres morphologischen Aussehens
aus derselben homogenen Substanz zu bestehen scheinen.

Die lim. ret. int. geht distalwirts nicht tiber das Oiliar-
epithel hinaus. Keine lim. ciliaris interna ist in diesem Alter
vorhanden. Die lim. ret. ext. andererseits setzt sich, wie schon
friher bemerkt worden ist, zwischen den beiden Schichten des
Ciliarepithels ununterbrochen fort. In der Hohe der ora ist sie
bemerkenswert dicker als distal dazu.

Die Linsenhohle ist noch vorhanden. Auf ihrer Kapsel sind
anscheinend nicht so viele Blutgefaisse zu finden als friiher. Die
Pupillenmembran ist noch unversehrt.

Der Zonularaum enthalt dieselben morphologischen Bestand-
teile, wie sie in 12 Stunden alten Exemplaren gefunden wurden.
Das Netzwerk ist jedoch weniger verworren und die Maschen
sind grésser. Die Gefiissmembran nimmt dieselbe relative Lage
in dem Zonularaum ein, ist aber diinner geworden und weniger
deutlich als eine Membran zu erkennen. Sie ist indessen aus
denselben spindelférmigen dunkel gefarbten Zellen und ihren Fasern
zusammengesetzt wie im vorigen Stadium. Unterbrechungen sind
éfters zu bemerken, und in diesen kann man mebhrere zarte
Faserchen sehen, die zur Linsenkapsel hinziehen. Diese Faserchen
sind in diesem Alter oft aus den grossen hellen, unrege]missigen
Zellen hervorgegangen, welche friiher schon beobachtet wurden.
Solche Zellen findet man noch in derselben Lage im Zonularaum,
nimlich entweder auf der Membran oder in dem freien Raum
zwischen ihr und dem Ciliarepithel. Daneben kann man noch
beobachten, dass einige der Zellen auf dem Ciliarepithel selbst
liegen. Beim Verfolgen der Netzwerkfasern, welche aus diesen
unregelmissigen Zellen kommen, bis zum Epithel bemerkt man
nunmehr mehrere solche Fasern bis zum Intercellularraum zwischen
benachbarten Zellen hinziehen, wihrend bei friiheren Exemplaren
alle diese Fasern an den spitzen Fortsiitzen der Epithelzellen
angeheftet waren. Ks haben freilich noch nicht viele Fasern
ihren apicalen Ansatz verloren; doch ist die Anzahl der Epithel-
zellen mit Apicalfortsitzen schon merklich verringert. Gelegent-
lich ist eine dickere Faser, welche aus den spindelférmigen Zellen

Die Entwicklung der Fasern der Zonula Zinnii. 281

der Membran hervorgeht, bis zum Epithel zu verfolgen, wie es
schon bei den 12 Stunden alten Exemplaren bemerkt wurde.
Diese Fortsatze werden indessen nur selten angetroffen und nur
bei Zellen, die in der Nahe der ora serrata und lim. ret. int. liegen.

Die dickeren Protoplasmafortsitze, welche friiher ohne Ver-
mittlung eines Zellkorpers direkt vom Epithel zur Gefassmembran
liefen, sind, gleichzeitig mit dem Diinnerwerden und dem teil-
weisen Verschwinden jenes letzteren Gebildes, nicht mehr vor-
handen. In diesem Alter ist keine Faser zu beobachten, welche
direkt und ohne Unterbrechung vom Ciliarepithel zur Linse geht.
Indessen werden viele Fasern gefunden, die, aus einer Zelle des
Zonularaumes stammend, direkt zum Epithel gehen, wahrend
ahnliche Fasern aus demselben Zellkérper in entgegengesetzter
Richtung zur Linsenkapsel laufen.

Die weisse Maus, 1l Tage alt.

In diesem Alter ist die Linsenkapsel von _ betrachtlicher
Dicke, und auf ihr verlaufen viele Blutgefasse. Diese sind auf
der proximalen Oberfliche zahlreicher als auf der distalen. Die
Linsenhohle ist infolge der Vereinigung der beiden Epithel-
schichten, welche ihre Wande bildeten, verschwunden, aber die
Pupillarmembran ist noch vorhanden.

Die Ciliarregion hat sich seit dem 5. Tage sehr entwickelt.
Die Ciliarfortsitze haben sich bedeutend vergréssert, erreichen
jedoch die Linse-noch nicht ganz. Jeder derselben enthalt einen
Kern von Mesenchymgewebe, das Gefasse mit vielen Blutkérperchen
umschliesst. Dicht an die aussere Oberfliche der éusseren Schicht
der Ciliarepithelzellen setzt sich eine Schicht sich verzweigender
verlingerter Mesenchymzellen an. LEinige ihrer Fortsiatze sind
zwischen diesen Epithelzellen zu verfolgen, wo sie sich mit der
homogenen Intercellularsubstanz verbinden und nicht weiter ver-
folgt werden kénnen. In keinem Falle kann man sie durch beide
Epithelschichten des Zonularaumes verfolgen.

Die ausseren Ciliarepithelzellen sind kurz, von saéulen- oder
wirfelformiger Gestalt und kérniger als jene der inneren Schicht.
Verfolgt man sie proximalwiarts, so findet man, dass sie an der
ora serrata in die Pigmentschicht der Netzhaut tibergehen. Wie
bei den jiingeren Exemplaren, kann auch bei diesem die lim.
ret. ext. als lim. cil. ext. ununterbrochen distalwirts verfolgt

282 W. M. Baldwin:

werden. An der ora serrata ist sie jedoch betrachtlich verdickt.
An keiner Stelle ist eine Verschiedenheit ihres Baues von dem
homogenen Gefiige der Intercellularsubstanz festzustellen, sowohl
bei der dusseren, wie bei der inneren Schicht der Epithelzellen,
mit denen sie sich direkt verbindet.

Weiterhin ist zu bemerken, dass die Intercellularsubstanz
zwischen jenen inneren Epithelzellen, welche in dem Gziirtel
zwischen den Ciliarfortsitzen und der ora serrata liegen, und
ebenso derjenigen im Raume zwischen henachbarten Fortsatzen
gleichmissig verdickt ist. Ein eingehendes Studium des Raumes
zwischen diesen Zellen an den auf dieses folgenden Stadien bis
zum 14. Tage fortschreitend, zeigt, dass das Dickenwachstum
dieser Substanz durch die ganze Linge der Zwischenraéume in
gleichmissiger Weise stattgefunden hat, d. h. die Zunahme zeigt
sich nicht zuerst an einem Ende des Raumes zwischen zwei
Zellen und schreitet dann allmihlich zum anderen Ende vor.
Andererseits. zeigt die Substanz zwischen benachbarten Epithel-
zellen, welche auf den Ciliarfortsitzen liegen, keine so merkliche
Zunahme an Dicke.

Ich habe oben erwihnt, dass sowohl die Intercellularsubstanz
zwischen den Zellen der ausseren Schicht, wie auch die zwischen
den Zellen. der inneren Schicht mit der lim. cil. ext. zusammen-
hingt; jedoch nur an sehr wenigen Stellen liegen diese Inter-
cellularsubstanzen in derselben Ebene und bilden so eine Scheide-
wand, welche die ganze Dicke des Ciliarepithels durchquert. Der
Bau der limitans und der der Intercellularsubstanzen scheint der-
selbe zu sein: ein zellen- und faserloses, homogenes Abscheidungs-
produkt, das sich dunkel und gleichmissig farbt.

Die Zellen der inneren Schicht sind siulenformig und linger
als die der fusseren Schicht. Sie haben ein verhaltnismassig
helles Protoplasma und einen zentral gelegenen, ovalen oder
unregelmissigen Kern. Mitosen sind in beiden Epithelschichten
nachzuweisen.

Die inneren Rander der inneren Epithelzellen, welche sich
auf den Ciliarfortsatzen finden, liegen offenbar in derselben Ebene.
Eine Anzahl sich dunkel farbender Fasern, die mehr oder weniger
eng verbunden erscheinen, liegen auf diesem Epithelrande und
sehen wie eine limitans ciliaris interna aus. Man kann indessen
an Exemplaren mit gelegentlich kiirzerer Epithelzelle, oder wo

Die Entwicklung der Fasern der Zonula Zinnil. 283

infolge der Praparation diese Fasern von der darunter liegenden
Epithelobertliche entfernt sind, leicht erkennen, dass die Rander
dieser Zellen nicht merklich verdickt sind, dass eine wirkliche
limitans ciliaris interna in Wirklichkeit nicht vorhanden ist.
Ferner erstreckt sich die lim. ret. int. nicht distalwarts tber
irgend einen Teil des Ciliarepithels hinweg.

Der Zonularaum erscheint in diesem Alter verhaltmassig
gross und ist vom Glaskérperraum durch die Membrana hyaloidea
getrennt. Diese Membran erscheint als ein diinnes, homogenes
' Gebilde; sie liegt proximal zur Linse und heftet sich an das
Epithel der ora serrata ganz ahnlich wie das Ende der lim. ret.
int. und unmittelbar distal von ihr. Bei genauer Betrachtung
sieht man, dass sie sich in eine Anzahl von Schichten auflost,
von denen jede sich an der Spitze einer Fpithelzelle spaltet und
sofort mit der Intercellularsubstanz verschmilzt. Auf der distalen
Oberfliche der Membran und dicht bei ihrer Befestigungsstelle
am Epithel sind viele unregelmassig gestaltete Zellen mit ovalen
oder unregelmissigen Kernen und mit mehreren Fortsatzen zu
bemerken. Einige derselben sind bis zu den Epithelzellen zu ver-
folgen, wahrend andere in entgegengesetzter Richtung nach der
Linse hin laufen, indem sie langs der distalen Oberflache der
Membran ziehen, mit welcher sie sich schliesslich verbinden.

Einige Blutgefasse sind in dem Raume noch vorhanden.
Die Membran, welche sie bei den jiingeren Exemplaren stiitzte,
ist jedoch als eine deutliche morphologische Einheit verschwunden.

Es ist wahr, dass die dunkel gefarbten, spindelférmigen
Zellen, welche dieses Gefiige durch ihre vereinigten dicken Fortsatze
friiher bildeten, noch in diesem Raume vorhanden sind; aber sie
sind nur noch auf die Gefiisswinde beschrankt. Ihre Fortsatze
laufen vereinzelt lings der Gefaisse und verschmelzen mit anderen
ahnlichen Fortsatzen, ohne jedoch eine stiitzende Membran zu bilden.

Die hellen, auslauferreichen Zellen der jiingeren Exemplare
sind noch ebenso zahlreich wie friiher vorhanden und nehmen
relativ dieselbe Lage ein wie auf der friiheren Stufe. Einige
davon scheinen jetzt nur zwei Fortsaitze auszusenden, von denen
der eine sich zur Linse, der andere zum Epithel hinzieht. Starke
Vergrésserung zeigt jedoch, dass dicht beim Epithel jede Faser sich
in viele feine Faserchen teilt, die sich alle entweder an eine Epithel-
zelle oder an die Intercellularsubstanz zwischen diesen Zellen heften.

284 W.M. Baldwin:

Tangentialschnitte belehren dariiber, dass viele dieser unregel-
massig gestalteten Zellen auf der Ciliarepitheloberflache liegen,
wo sie anastomosierende Fasern abgeben, die das Augeninnere
umschliessen. Diese Fasern, sowie auch jene, die ich oben schon
erwihnte, und die vom Epithel zur Linse ziehen, nehmen, wenn
sie dicht am Epithel der Ciliarfortsétze angeheftet sind, das
Aussehen einer limitans dieser Zellen an. Keine der Epithel-
zellen der Ciliarfortsitze jedoch erhalt Fasern. Die letzteren
gehen ununterbrochen weiter, ohne mit diesen Zellen in strukturale
Verbindung zu treten. Einige der nach der Linse ziehenden —
Fasern vereinigen sich mit einem Bipolarzellenfortsatz, der auf
einer Blutgefasswand liegt; aber keine Faser tritt schliesslich
mit den Endothelzellen der Gefisswande selbst in Verbindung.

Kin eingehendes Studium der Anheftungsweise der Fasern
an das Ciliarepithel zeigt, dass neben den beiden oben erwihnten
Arten der Verbindung, namlich an die apicalen Fortsatze und
an die Intercellularriume der Epithelien noch die folgenden hin-
zutreten. Manche Fasern, die an den Apicalfortsiitzen der Epithel-
zellen inserieren, haben verschiedene Beziehung zu dem Cuticular-
rande dieser Zellen und zu der angrenzenden Intercellularsubstanz.
In einigen Fallen ist namentlich bei den jiingeren Exemplaren
haufiger zu bemerken, dass die zarten Fasern an den Apical-
fortsitzen plétzlich aufhoren. In anderen sind die Cuticular-
rander der Epithelfortsitze verdickt und ziehen sich langs der
angehefteten Fasern nach der Linse hin. Wiederum kann die
Verdickung der Cuticularrander auf eine Seite des Fortsatzes
beschrankt sein, wahrend in noch anderen Fallen die Cuticula
auf einer Seite des Fortsatzes nur auf eine kurze Strecke der
Entfernung von der Intercellularsubstanz bis zur angehefteten
Faser verdickt ist. In keinem Falle aber findet man einen ver-
dickten Cuticularrand nur auf die Ansatzstelle der Faser beschrankt
und ohne Verbindung mit der benachbarten Intercellularsubstanz.
Wo die Fasern in diesem Alter sich direkt mit der Intercellular-
substanz verbinden, sind sie durch diese sich dunkel farbende
Masse nicht weiter zu verfolgen.

Die weisse Maus, 14 Tage alt.

In diesem Alter hat das Auge annahernd seine endgiltige
Beschaffenheit erreicht. Die Augenlider sind offen, und die

Die Entwicklung der Fasern der Zonula Zinnii. 285

Pupillarmembran ist verschwunden. Im Aquator der Linse sind
indessen noch einige Blutgefaisse zu bemerken. Die membrana
hyaloidea, welche den Glaskérperraum vom Zonularaum trennt,
ist dicker und dunkler gefirbt, aber ihre Beziehungen und Ver-
bindungen sind dieselben wie bei den Exemplaren von 11 Tagen.
Der Zonularaum ist von den zahlreichen Zonulafasern ein-
genommen, welche gewohnlich direkt vom Ciliarepithel zur Linsen-
kapsel ziehen. Sie sind an diese entweder Aquatorial, oder wie
bei einigen wenigen, distal zu dieser Region angeheftet. Die
meisten setzen sich jedoch an jenen Teil der Linsenkapsel an,
welcher sich vom Aquator zu dem proximalen Pol erstreckt.
Einige der Fasern dieser letzten Gruppe heften sich an die distale
Oberflache der membrana hyaloidea. Man kann diese Fasern
eine kurze Strecke auf der Linsenkapsel verfolgen; dann ver-
einigen sie sich anscheinend mit ihr und sind nicht weiter zu
verfolgen. Andere dagegen vereinigen sich mit den Fortsatzen
aus den Bipolarzellen, welche sich auf den Gefassen langs der
Linsenkapsel befinden.

Die Anheftung der Zonulafasern an die pars ciliaris retinae
ist wie bei der 11 Tage alten Maus auf die Teile dieses Epithels,
welche zwischen den Ciliarfortsitzen und der ora serrata liegen,
und auch auf die Vertiefungen zwischen benachbarten Fortsatzen
beschrankt. Keine dieser Fasern ist an dem Epithel auf den
Ciliarfortsatzen befestigt. Jede Faser sitzt entweder an der Spitze
eines Epithelzellenfortsatzes oder an der Intercellularsubstanz
zwischen aneinander grenzenden Zellen an. Es sind jedoch auch
verschiedene spitze Epithelzellen zu beobachten, die keine Faser-
ansitze haben. Im ganzen sind weniger spitze Zellen vorhanden
als bei den jiingeren Exemplaren. Zu erwihnen wire noch, dass
sie bei den alteren Exemplaren weniger oft auf den distalen
Teilen des Ciliarepithels angetroffen werden, .aber dass sie selbst
im Alter auf den Schnitten niemals ganz fehlen.

Bei eingehendem Studium der Intercellularsubstanz ist leicht
zu sehen, dass das, was als direkte Verlangerungen der Zonula-
fasern erscheint, sich als deutliche faserartige Bander erkennen
lasst, die von der homogenen Intercellularsubstanz, in der sie
liegen, sehr verschieden sind, und welche sich dunkel firben.
Sie kénnen nach der lim. cil. ext. hin verfolgt werden, ohne
diese jedoch ganz zu erreichen. Bei einigen Schnitten, wo das

286 W.M. Baldwin:

Messer gerade neben der flachen Oberfliche einer Schicht Inter-
cellularsubstanz eindrang, waren diese Fasern am besten als sich
dunkel farbende Faden zu sehen, die sich nach der 4Ausseren
Zellschicht hinziehen. In keinem Falle indessen liegen diese
Faserchen, wie Tangentialschnitte zeigen, innerhalb der Epithel-
zellkorper.

Nicht alle Zonulafasern gehen ununterbrochen zur Linse,
da yiele verlangerte, spindelformige Zellkérper ihren Lauf unter-
brechen. Diese Zellen haben einen oyalen oder unregelmissigen
Kern, der von reichlichem und in manchen Fallen dunkel ge-
firbtem Protoplasma umgeben ist. Sie geben in der Regel zwei
Fasern ab, yon denen eine zur Linse, die andere zum Ciliar-
epithel geht; hier teilt sich jede in eine Anzahl feiner Faserchen
und erlangt eine Verbindung mit den Epithelzellen ganz ahnlich
wie andere Zonulafasern, in deren Verlauf keine Zellen nachzu-
weisen sind. Zellen sind durch den ganzen Zonularaum zerstreut ;
sie liegen aber gewohnlich naher zum Epithel als zur Linse. Kinige
sind dicht an den Ciliarepithelzellen zu finden und senden Fort-
sitze aus, die sich mit Fortsitzen aus ahnlich gelegenen Zellen
vereinigen und das Innere des Augapfels umkreisen.

Gelegentlich ist auch eine Zelle auf einer Zonulafaser zu
bemerken; in welcher nur die Umrisse des Kernes und des Zell-
kérpers vorhanden sind. Die Faserfortsitze solcher ,,Schatten-
zellen* sind trotzdem gut erhalten und dunkel gefarbt.

Das Ciliarepithel zeigt dieselben Besonderheiten wie bei den
11 Tage alten Mausen; ausgenommen, dass es sich in ver-
schiedener Hinsicht in einem vorgeschritteneren Entwicklungs-
stadium befindet. Fortsitze von Mesenchymzellen nahe dem musc.
ciliaris treten in die Intercellularsubstanz der dusseren Epithel-
schicht ein; aber keiner dieser Ausliufer geht hindurch bis in
den Zonularaum. Die Intercellularsubstanz der inneren Epithel-
schicht ist an jenen Stellen, wo Zonulafasern ansetzen, im Ver-
gleiche zu der auf den Ciliarfortsitzen verdickt.

Das Protoplasma mehrerer ciliarer Epithelzellen der ausseren
Schicht dringt nach innen zu in den Raum zwischen angrenzenden,
dariiber liegenden Zellen der inneren Epithelschicht ein. Man
findet jedoch nicht, dass die Zonulafasern mit solchen Ver-
langerungen zusammenhingen. Auf keinem Teile des Ciliar-
epithels ist eine lim. cil. int. vorhanden. Ich muss auch hier

Die Entwicklung der Fasern der Zonula Zinnii. 287

wieder wie bei den 11 Tage alten Maéusen erwahnen, dass das
Aussehen einer solchen limitans dadurch hervorgerufen wird, dass
mehrere Zonulafasern sich tiber die Oberfliche von Zellen auf
den Ciliarfortsitzen hinziehen; die lim. ret. int. dagegen erstreckt
sich nicht iiber das Ciliarepithel.

Uberblicken wir nun kurz die ganze Reihe der untersuchten
Mause, so ergibt sich folgendes Bild der aufeinanderfolgenden
Entwicklungsstadien :

Vom ersten Tage der Geburt an sind die lim. ret. int. und
die lim. ret. ext. als deutliche Membranen vorhanden. Die membrana
limitans interna ist indes die stirkere von beiden, und ihre Starke
wird noch dadurch betrachtlich vergréssert, dass sich ihrer inneren
Oberflaiche zahlreiche Fasern anlegen, die von dem urspriinglichen
Glaskérpergewebe herstammen. Diese Membran kann an ihrem
iiussersten Ende nur bis zur ora serrata verfolgt werden, die
schon als die Verbindung zwischen der pars ciliaris retinae und
der pars optica retinae bezeichnet wurde. Dort endigt sie, indem
sie sich in verschiedene Lamellen teilt, wovon jede sich an eine
Epithelzelle der ora serrata anlegt. Dagegen erstreckt sich die
lim. ret. ext. von Anfang an an ihrem distalen Ende zwischen die
zwei Schichten von Ciliarepithelzellen als eine ununterbrochene
Membran, die limitans ciliaris externa. Die nachstfolgenden Tage
hindurch nimmt die lim. retinae int. sowohl an Substanz wie aucb
an Anzahl der Lameilen zu, in die sie sich am fussersten Ende
teilt. Die Intercellularsubstanz, mit der die limitans direkt zu-
sammenhangt, wichst in einem entsprechenden Verhaltnis an
Starke und ist schon am 6. Tag in merklichen Gegensatz zu der
zwischen anderen benachbarten Zellen getreten. Ihr Zusammen-
hang mit der lim. ret. ext. ist ebenfalls klar ersichtlich, und es
muss ferner bemerkt werden, dass letztere an dieser Stelle schon
betrachtlich dicker geworden ist.

Schon am ersten Tage sind die Ciliarkérperfortsatze auf-
getreten, jeder mit dem zweischichtigen Ciliarepithel und einem
Kern aus Mesenchymgewebe, das Blutgefisse mit ihren Blut-
kérperchen enthalt. Die Ciliarfortsitze wachsen allmahlich an
Grosse und Zahl bis zum 14. Tag, wo sie ihre grésste Ent-
wicklung erreicht haben. Es muss bemerkt werden, dass in den
spiteren Entwicklungsstufen einige dieser Fortsatze verastelt
sind. Ferner beginnt die Intercellularsubstanz, die zwischen ge-

288 W. M. Baldwin:

wissen Epithelzellen der inneren Schicht liegt, vom 7. Tage an
sich allmahlich zu verstarken. Dieses Wachstum findet in einem
einheitlichen Verhaltnis in der ganzen Lange der Zwischenraume
angrenzender Zellen statt. Es muss noch hinzugefiigt werden,
dass die Teile des Epithels, wo dieses Wachstum stattfindet, sich
auf die ringformige Zone zwischen den Ciliarkérperfortsatzen und
der ora serrata, sowie auf die Zwischenréume zwischen den
Ciliarfortsitzen beschranken. Gleichzeitig verstaérkt sich auch
die lim. cil. ext., soweit sie in dieser Gegend anliegt. Eine Unter-
scheidung der Zellen der Ausseren Ciliarschicht von denen der
inneren ist von Anfang an méglich wegen ihrer morphologischen
Eigentiimlichkeiten. Die Zellen der inneren Schicht sind in den
jiingsten untersuchten Stadien fast alle spitz, und auf jeder Spitze
liegt eine Faser, die von dem den Zonularaum ausfiillenden Netz-
gewebe herkommt. Mit fortschreitender Entwicklung werden die
spitzen Zellen, die auf den Ciliarkérperfortsatzen liegen, allmihlich
in Zellen umgebildet, die eine flache, gegen die Linse gerichtete
Oberfliche darbieten. Diese .Verinderung hat sich schon am
5. Tag vollzogen. Die spitzen Zellen sind demgemass beschrankt
auf die zwischen diesen Ciliarkérperfortsitzen liegenden Taler
und auf die Zone, die sich zwischen letzteren und der ora serrata
ausdehnt. Aber selbst in diesen Gegenden nehmen diese Zellen,
indem sie der Verschiebung der Zonulafasern von einer apicalen
Insertion zu einer intercellularen sich anpassen, an Zahl ab.
Indessen verschwinden sie nie ganz aus einer Schnittserie, da
sogar bei der ausgewachsenen Maus noch viele solcher Zellen
gefunden werden.

Die Umbildung solcher spitzen Zellen und die Verainderung
in der Insertion der Zonulafasern erfolgt gleichzeitig mit dem
Wachstum der Intercellularsubstanz, die zwischen den Epithel-
zellen der Zonulagegend liegt. Zwischen dem 8. und dem 11.
Tage kann man diese Verainderungen am besten wahrnehmen.
Hinzuzufiigen ist, dass durch die ganze Serie Epithelzellen nach-
gewiesen werden kénnen, die einen spitzen Fortsatz haben, der
aber mit keiner Zonulafaser verbunden ist.

Der Glaskérperraum ist am Anfang der Serie mit der ent-
sprechenden Glaskérpersubstanz angefiillt, die aus einem losen Netz-
werk von Faserchen besteht. Diese kommen von verastelten oder
bipolaren Zellen her, welche die zahlreichen Blutgefasse umgeben.

Die Entwicklung der Fasern der Zonula Zinnii. 289

Von Anfang an wird auf diesen Zellen und Fasern ein
kérniger Niederschlag bemerkt. Keine Scheidewand trennt in
den friiheren Entwicklungsstufen den Glaskérperraum von dem
eigentlichen Zonularaum. Die Glaskérpersubstanz wird auch in
dem letzteren Raum gefunden und weist dieselben Elemente in
ihrer Zusammensetzung und dieselbe allgemeine morphologische
Gestalt auf. Es erfolgt sodann eine allmahliche Verminderung
in der Zahl dieser Elemente bis zum 5. Tag, wo der Raum von
kérnigem Niederschlag frei ist. Das Netzwerk ist ebenso in
diesem Alter weniger deutlich, aber die Blutgefasse und die sie
tragenden Zellen und Fasern sind noch vorhanden und kénnen
sogar bis zum 27. Tag nachgewiesen werden, allerdings an Zahl
bedeutend verringert.

Die Membrana hyaloidea erscheint nach dem Verschwinden
der kérnigen Substanz aus dem Zonularaum und hat schon am
10. Tag die morphologischen Eigentiimlichkeiten des fertigen Zu-
standes nahezu erreicht.

Neben den urspriinglichen Bestandteilen der Glaskérper-
substanz, die man im Zonularaum findet, ist dort noch ein Zell-
typus vorhanden, der nicht im Glaskérperraum vertreten ist. Es
sind dies helle, grosse, schwach gefarbte Zelien mit einem grossen
ovalen oder unregelmassigen Kern, reichlichem Protoplasma und
einem unregelmiassig gestalteten Zellkérper. Zellen von diesem
Typus liegen entweder auf der Gefiissmembran oder frei im
Zonulagebiet. Jede Zelle gibt sehr viel feine, helle Protoplasma-
fortsatze ab. Diese Fortsiitze verzweigen sich, anastomosieren
und verschmelzen sehr oft und bilden so ein wirres Netzwerk
zwischen der Blutgefissmembran und der ganzen Lange des
Ciliarepithels. Die einzelnen Fasern, welche solch ein Netzwerk
bilden, heften sich schliesslich an die Spitze einer Ciliarepithel-
zelle an.

Diese Zellen nehmen an den allmahlichen regressiven Ver-
anderungen, welchen die Bestandteile der Glaskérpersubstanz
unterworfen sind, nicht teil, sondern bleiben bestehen, und nehmen
womoglich mit der fortschreitenden Vergrésserung des Zonula-
raumes verhaltnismissig an Zahl zu. Gleichzeitig aber mit der
schnellen Entwicklung der Ciliarfortsitze und mit den iiberein-
stimmenden morphologischen Veranderungen in den Epithelzellen,
welche sie bedecken, wird das aus den Zellen heryorgehende

I9Q W. M. Baldwin:

Netzwerk in seiner Lage eingeschrankt. Spater heftet es sich .
nur an das Epithel des Strahlenkranzes und an die Vertiefungen.
Zu gleicher Zeit wird es entsprechend den regressiven Ver-
inderungen der Gefasse und der daran gelegenen Bipolarzellen-
fasern weniger verworren. Auch aus einem anderen Grunde
noch anastomosieren, gleichzeitig mit der Vergrésserung des
(Juerdurchmessers des Zonularaumes, die Fasern aus den hellen
Zellen viel weniger oft. Sie werden betrachtlich langer und
dicker, dunkler gefarbt und verschmelzen in dem Augenblick, wo
sie den Zellkorper verlassen. Diese Vereinigung hat die Wirkung,
solche helle Zellen bipolar erscheinen zu lassen, wobei eine Faser
sich nach der Linse, die andere nach dem Epithel hinzieht.
Wenn indessen der letztere Fortsatz bis zum Ciliarepithel verfolgt
wird, so stellt sich heraus, dass er sich in eine Anzahl ausser-
ordentlich feiner Fiaserchen zerteilt, welche in ihrem morpho-
logischen Charakter und ihrem Verhalten gegeniiber der Farbung
genau den zarten Faserchen gleichen, welche das Netzwerk bei
den jiingeren Exemplaren bildeten.

Endlich wird das Netzwerk allmahlich durch die Zonula-
fasern ersetzt, welche eine direkte Richtung vom Epithel zur
Linse einschlagen. Beinahe jede Faser ist zuerst von einem
Zellkérper unterbrochen. Im vorgeschrittenen Stadium vermehrt
sich die Zahl der Zonulafasern, welche keine Zellkorper tragen,
allmahlich. Bei den ausgewachsenen Exemplaren sind sehr wenige
solcher Zellkérper zu bemerken. Aber sobald diese verschwinden,
treten die ,Schattenzellen“ auf. Letztere haben sehr deutliche
und anscheinend gut erhaltene Zonulafaserfortsatze, jedoch nur
den Umriss eines Kernes und eines Zellkorpers. Ich kann daraus
nur schliessen, dass diese ,Schattenzellen“ entartete, helle Zell-
korper sind, in welchen die Faserfortsitze schliesslich als Zonula-
fasern des fertigen Auges bestehen bleiben.

Seit dem ersten Bericht von Zinn im Jahre 1775 sind
von den Forschern viele und verschiedene Ansichten iiber den
Ursprung, die Bedeutung und die letzten morphologischen Verhialt-
nisse der Zonulafasern aufgestellt worden. Collins (1891)
betrachtete sie als Fortsitze der Linsenzellen, welche sich zu
den Ciliarepithelzellen erstreckten und schliesslich mit diesen
vereinigten, eine Ansicht, die heute von den Gelehrten kaum
anerkannt wird.

Die Entwicklung der Fasern der Zonula Zinnii. 291

Eine Anzahl von Forschern haben die Fasern aus dem ur-
spriinglichen Glaskérpergewebe abgeleitet.1) Diesen Standpunkt
nehmen ein: Lieberkiihn, Angelucci, Loewe, Schwalbe,
Haensell, Iwanoff, Salzmann, de Waele, Retzius und
von Lenhossék. Die Arbeit des zuletzt genannten Forschers
wurde auch an Saugetieren, einschliesslich des Menschen, aus-
gefiihrt, griindete sich jedoch grésstenteils auf ein Studium von
Hiihnerembryonen vom 4. bruttage an. Er sah die Fasern frei
in der distalen Verlingerung des Glaskérperraumes zwischen der
Linse und dem Ciliarepithel liegen.. Sie bildeten zuerst ein ver-
zweigtes Netzwerk, ahnlich dem urspriinglichen, anderswo zu
findenden Glaskérpernetzwerk, und lésen sich, unbeeinflusst vom
Zusammenhange mit Zellen des urspriinglichen Glaskérpers. der
Linse oder des Ciliarepithels, allmahlich in deutliche verzweigte
Fasern auf, die von der Linse direkt zum Ciliarepithel laufen,
‘ wo sie sich zuletzt mit der Intercellularsubstanz jener Region
verbinden. Er sah zuerst einen deutlichen Zwischenraum, welcher
die Zonulafasern vom Ciliarepithel trennte, der aber spater von
den Fasern iiberbriickt wurde.

Lenhosséks Entdeckungen mégen den Ursprung dieser
Fasern bei Végeln zeigen. Meine eigenen Resultate lassen mich
jedoch nach dem, was ich gefunden habe, glauben, dass die Ent-
wicklung bei Siugetieren anders verlauft. In diesem Zusammen-
hang ist es interessant, dass Rabl. der an Menschen, Schafen,
Schweinen, Végeln, Selachiern und Amphibien arbeitete, Ergebnisse
berichtete, welche zeigten, dass die Entwicklung der Fasern beim
Hiihnchen von der bei anderen Tierformen verschieden ist. Bis
wir demgemass den infolge von Schrumpfung der Praparate ent-
standenen Fehler ausgeschaltet haben, der in einer Anzahl ver-
offentlichter Zeichnungen zutage tritt, und der den yon Lenhossék
gesehenen Raum zwischen den Fasern und dem Epithel erklaren
mag, miissen wir etwas skeptisch sein gegeniiber der Fahigkeit
der Fasern, sich frei von jeder Zellentitigkeit so zu organisieren,
arrangieren und anzusetzen, wie der Verfasser es beschrieben
hat. Und wir diirfen nicht den zweiten Fehler begehen, zu
folgern, dass das etwa fiir das Hiihnchen Richtige notwendiger-
weise auch fiir Siugetiere gelten miisse.

) Auf die Controversen iiber die erste Entstehung des Glaskirpers
wird hier nicht eingegangen.

.

292 W. M. Baldwin:

Zinn, Cloquet, Dessauer, Claeys, Czermak,
Topolowski, Collius, Agababow, Terrien und Metzner
schlossen, dass die Zonulafasern aus dem Ciliarepithel entstiinden.
Schoen sah sie fiir Protoplasmafortsitze an, die aus den inneren
Epithelzellen erwiichsen. O. Schultze, Sbhordane, Fischel
(der am. Salamanderauge arbeitete), Rabl und Addario waren
derselben Meinung. Damianoff indessen betrachtete diese
Fasern als ein Ausscheidungsprodukt dieser Zellen. Schoen
bemerkte iiberdies, dass jede Epithelzelle eine Faser hergab, die
durch Verbindung mit ihren Nachbarn eine wirkliche Zonulafaser
bildete. Von Spee beobachtete ihren Ansatz an spitze Epithel-
zellen und schloss daraus, dass sie in Wirklichkeit eine Art von
Cuticularprodukt dieser Zellen seien. Salzmann und yon
Ebner teilten diese Ansicht; letzterer erklarte dazu, er kénne
sehen, dass einige der Fasern in die Epithelzellen eindrangen.
Kélliker behauptete ebenfalls den Epithelstandpunkt die Fasern
betreffend, indem er annahm, dass sie in genetischer Beziehung
zu den Fasern des Glaskérpers stinden, trotz der tatsichlichen
Verschiedenheit ihrer chemischen Reaktionen.

Will man durch Ausschluss zu einem Beweise kommen, auf
Grund der oben erwahnten Arbeiten, dass die Zonulafasern
wirklich Auswiichse der inneren Ciliarepithelzellen darstellen, so
gibt es in bezug auf den Vorgang, durch welchen sie zu ihrer
endgiiltigen Lage gelangen, nur zwei Méglichkeiten.

Die erste ist die, dass die Verbindung zwischen den
Kpithelzellen und der Linse in einer fritheren Periode, als diese
Teile einander beriihrten, entstand, und dass als Resultat des
Auftretens des Zonularaumes und der allmahlichen Vergrésserung
seines Querdurchmessers diese Fasern, welche ihre Ansatzstelle
an der Linse noch behaupteten, linger und langer wurden, bis
schliesslich der endgiiltige Zustand erreicht war.

Diese Annahme kann in zwei Hauptpunkten kritisiert
werden. Erstens: So viel ich weiss, hat noch kein Autor bei
Saugetieren eine Form beschrieben, in welcher selbst in den
friiheren Stadien die Linse und die Ciliarregion jemals in direkter
Beriihrung miteinander gestanden hatten. Eine Schicht von
Mesenchymgewebe mit Blutgefissen trennt diese beiden Gebilde
von Anfang an. Zweitens (hier kann ich nur von meinen Be-
obachtungen an der weissen Maus sprechen): Die von mir be-

Die Entwicklung der Fasern der Zonula Zinnii. 23

merkten spitzen Zellen, welche Protoplasmafortsitze abgeben,
die ununterbrochen zur Linse laufen, waren jene auf den Ciliar-
fortsitzen, wo spiter keine Zonulafasern angeheftet sind. Drittens:
An jenen Flichen, wo die endgiiltigen Fasern angeheftet sind,
habe ich auf den friiheren Stufen der Entwicklung keine Proto-
plasmafortsitze finden kénnen, die direkt und ununterbrochen zur
Linse gehen.

Die Zonulafasern miissen sich demgemiss in einer spiteren
Periode, wenn ein Raum zwischen der Linse und dem Ciliarepithel
vorhanden ist, entwickelt haben. Wir kénnen unsere Aufmerk-
samkeit daher auf die zweite Annahme lenken.

Diese ist kurz folgende: um einen Beweis von dem Epithel-
ursprung der Fasern richtig zu begriinden hinsichtlich der un-
zweifelhaften Tatsache, dass der Raum, durch welchen sie laufen,
schon von vielen Mesenchymzellen und Fasern, die selbst einen
Epithelansatz haben, eingenommen ist, miissten wir notwendiger-
weise diese Fortsatze auf verschiedenen Stufen des genetischen
Fortschreitens gesehen haben, wie sie aus dem Epithel hervor-
wachsen und zur Linse fortschreiten, bis sie sich spiter dort an-
setzen. Ein solcher Beweis fehlt in der Arbeit der erwahnten
Forscher. Demgegeniiber habe ich in meinen Schnitten mehrere
l'asern bemerkt, die eine kurze Strecke zur Linse hin liefen und
dann plétzlich endeten. Diese Fasern waren immer von anderen
begleitet, welche die ganze Strecke zur Linse durchliefen. Selbst
mit den besten Linsen, die mir zu Gebote standen, und bei
ungefahr 2000facher linearer Vergrésserung habe ich in keinem
Falle bestimmen kénnen, ob das Ende der Fasern das _ spitze
Knde einer wachsenden Faser oder das durch das Messer ab-
geschnittene Ende einer Faser war, welche sich ein wenig unter
der Ebene ihrer Nachbarn befand. Auch heben Serienschnitte
trotz sorgfaltigster Ausfiihrung die Schwierigkeiten nicht auf.
Das Haupthindernis ist die Orientierung dieser Fasern beim
Ubergang von einem Schnitte zum nachsten der Serie. Und
wenn man bedenkt, dass sie in einem hellen Raume liegen, ver-
haltnismassig weit entfernt von festen Punkten, die zur Lage-
bestimmung dienen kénnten; wenn man auch die Leichtigkeit
bedenkt, wie solche Fortsetzungen durch die Priparation ver-
loren gehen oder verschoben werden, selbst bei den am sorg-
faltigsten behandelten Exemplaren: so muss man gestehen, dass

294 W. M. Baldwin:

hierin eine Schwierigkeit fiir unser Studium liegt, die bis jetzt
fast uniiberwindlich ist.

Uberdies kann ich bei weiterer Kritik dieser zweiten An-
nahme hinzufiigen, dass meine eigene Arbeit und die vieler anderer
Forscher, iiber deren Ergebnisse ich spater ausfiihrlicher reden
werde, zeigen, dass die Zonulafasern schliesslich einen Inter-
cellularansatz haben und nicht an einen Apicalfortsatz auf dem
Ciliarepithel ansetzen. Gerade wie diese Verainderung des An-
satzes erfolgt, und durch welche verschiedenen Vorgange sie
zustande gekommen ist, das hat keiner der Verfechter des
Epithelursprungs erklaren kénnen, wenn sie auch den spateren
Intercellularansatz ebenfalls bemerkt haben.

Wie koénnen wir weiter das Vorhandensein von Zellen mit
deutlichem Zellumriss und Zellkernen auf den Zonulafasern und
dem Ciliarepithel erklaren bei Mausen, die beinahe voll aus-
gewachsen sind? Lenhossék und andere haben runde oder
unregelmiissige Zellen mit einem deutlichen unregelmassigen Kern
in solechen Lagen beobachtet und sie fiir Leukocyten erklart. Ich
habe diese Zellen ebenfalls gesehen und bin zu demselben Schluss
gekommen. Aber die Zellen, auf die ich mich besonders beziehe,
geben Zweige ab, die zur Linse und zum Epithel laufen. Wolfrum
sah solche Zellen in seinen Exemplaren. Nussbaum beobachtete
sie vor ihm beim Kaninchen. Ich habe sie bei jedem Schnitt
durch die ganze Reihe der weissen Mause gefunden und sie
iiberdies auch im Auge des erwachsenen Menschen bemerkt.

Das Fehlen oder Vorhandensein einer wirklichen Grenz-
membran auf dem freien Rande der Ciliarepithelzellen der inneren
Schicht ist von mehreren Forschern als Beweis fiir oder gegen
den Lauf der Zonulafasern zu einem tieferen Ansatz in dem
Epithel dieser Region angefiihrt worden. Lenhossék z. B.
glaubte an das Vorhandensein einer wirklichen limitans ciliaris
interna, deren Funktion es sei, die Zonulafasern mit dem darunter-
liegenden Intercellulargewebe, mit dem sie direkt zusammenhangt,
in Beziehung zu bringen. Diese Membran ist nach ibm ununter-
brochen. Darin fand er einen Beweis gegen das tiefere Vordringen
der Zonulafasern. Czermak hielt die limitans fiir eine hyaline
Struktur, welche mit dem Glaskérper in direktem Zusammen-
hange steht. Aus dieser Schicht stammten die Zonulafasern.
Topolowsky bestatigt diese Ansicht. Fischel sah die limitans

Die Entwicklung der Fasern der Zonula Zinnii. 295

als die direkte distale Verlangerung der lim. ret. int. an, Salzmann
und vy. Ebner konnten die Zonulafasern nur bis zur limitans
verfolgen. Mawas glaubte an das Vorhandensein einer limitans,
stellte aber fest, dass dies kein Hindernis fiir das tiefere Kin-
dringen der Zonulafasern sei, von denen einige eine Strecke weit
in der darunter liegenden Intercellularsubstanz zu verfolgen
seien. Der letztere Forscher betrachtete ferner die lim. cil. int.
nicht als wirkliche Membran, sondern nur als ein exoplasmatisches
Produkt der angrenzenden Zellen.

Ich habe oben von meinen Ergebnissen in bezug auf die
lim. cil. int. gesprochen. Bei der Maus ist sie sicher auf keiner
Stufe der Entwicklung vorhanden. Indessen erzeugt der Lauf
der Zonulafasern und anderer Zellgewebsfasern quer iiber die
Epitheloberflichen an mehreren Stellen das Bild einer Grenz-
membran.

Von Claeys wurde ein interessanter Gedanke betreffs
einiger analogen und morphologischen Eigenschaften des Ciliar-
epithels und der eigentlichen Retina 1886 verétientlicht. Er
nahm an, dass dieses zweischichtige Epithel ein System von
Stiitzzellen und -fasern besisse, ahnlich den Miillerschen Fasern
der Retina, und dass die Zonulafasern nur die inneren Ver-
langerungen solcher Fasern waren. Diese Ansicht fand spater
einen Verfechter in Terrien. Letzterer beschrankte jedoch die
Zonulafasern nicht auf diese Stiitzzellen, da er einige durch die
ganze Dicke der Ciliarepithelschichten bis zum iusseren Mesenchym-
gewebe des Ciliarkérpers verfolgen konnte. Neuerdings unter-
stiitzte Metzner diese Theorie und gab an, die Zonulafasern
bis zur Scheide des m. ciliaris verfolgt zu haben.

Im Jahre 1906 ging Toufesco so weit, zu behaupten,
dass die Zonulafasern aus elastischem Gewebe bestinden, dass sie
beide Ciliarschichten durchdringen und so eine direkte Verbindung
mit ahnlichem Gewebe in der Aderhaut des Augapfels herstellten.

Ich habe schon friiher berichtet, dass ich bei einigen meiner
Exemplare Mesenchymzellen bemerken konnte, die an der ausseren
Oberflache der ausseren Epithelzellschicht angeheftet waren, und
die gelegentlich Fortsitze abgaben, welche nach innen zu in die
Intercellularsubstanz zwischen diesen Zellen eindrangen. Ich
konnte sie jedoch nur eine sehr kurze Strecke zwischen diesen
Zellen verfolgen, da sie morphologische Eigenschaften und Farb-

296 W. M. Baldwin:

barkeit besitzen wie die homogene Substanz, in der sie liegen.
Daher bin ich auch nicht imstande, die Beobachtungen dieser
Autoren zu bestatigen. Wenn ich nach einigen der veréffentlichten
Zeichnungen urteile, kann ich nur schliessen, dass, wie ich bei
ahnlichen Erscheinungen unter dem Mikroskop sah, diese Forscher
mit Schnitten arbeiteten, die sehr schrag angelegt waren.

Wenden wir unsere Aufmerksamkeit zunichst auf die Inter-
cellularsubstanz, die in den Zonulaflichen des Ciliarepithels vor-
handen ist, und welche die yon vielen Forschern bemerkten
Zonulafaserverlingerungen enthalt, so finden wir, dass in Ver-
bindung mit dieser Sache N. van der Stricht, Leboucg
und O. yan der Stricht, die tiber die limitans des Gehor-,
des Geruchs- und des Sehepithels arbeiteten, zu dem Schlusse
gelangten, diese homogenen Membranen seien nicht wirkliche
Membranen, sondern nur ein strukturloser intercellularer Kitt. Bei
meinen eigenen Serienschnitten kann ich, wie ich schon konstatiert
habe, keine geformten Gewebsbestandteile in der limitans be-
merken. (Kine Beschrankung dieser Behauptung werde ich spiter
geben.) Ihr morphologisches Aussehen und ibr Verhalten bei
der Farbung ist dem der ganzen Intercellularsubstanz des Ciliar-
epithels, mit der sie in direktem Zusammenhange zu _ stehen
scheint, vollig gleich.

Wenn wir in dem Falle dieser Intercellularsubstanz annehmen,
dass sie als eine Art exoplasmatischen Produkts der benachbarten
Zellen gebildet ist, wobei eine Zelle nicht mehr als die andere
zu ihrer Dicke beitrigt, — und es gibt in der Literatur, wie ich
glaube, nichts, was dieser Ansicht widerstreitet — warum sollten
wir so weit gehen, zu behaupten, dass diese lim. ext., die allem
Anscheine nach aus demselben Stoff zusammengesetzt ist, mehr
aus den Zellen der inneren Ciliarschicht hervorgeht, als aus
denen der ausseren? Dabei diirfen wir nicht vergessen, dass
die Hypothese von der Analogie der inneren Ciliarepithelzellen
und derjenigen der Stiitzzellen der Retina noch nicht sicher
gestellt ist. Wir kénnen daher gerechterweise auch nicht ver-
muten, dass die lim. cil. ext. ein Derivat von Zellkérpern ist, die
nach innen von ihr liegen, wie es anscheinend bei der lim. ret.
ext. der Fall ist.

Mawas z. B. nimmt den Standpunkt ein, dass die lim. ext.
allein von den Epithelzellen der inneren Schicht gebildet ist.

x

Die Entwicklung der Fasern der Zonula Zinnii. 29

Er gibt indessen in seiner Arbeit nicht geniigende Beweise fiir
die Richtigkeit dieser Annahme. Wenn wir dann im Laufe der
Entwicklung beobachten, wie die Intercellularsubstanz im Zonula-
gebiet allmahlich an Dicke zunimmt, aber nicht bemerken, dass
diese Zunahme zuerst an einem Ende eines Intercellularraumes
auftritt und allmahlich zum anderen fortschreitet, sondern jm
Gegenteil in gleichmassiger Weise auf der ganzen Lange des
Zwischenraumes yor sich geht: dann haben wir keinen Grund
fiir die Annahme, dass diese Zunahme mehr den Zellen der
ausseren als denen der inneren Schicht zu yerdanken ist. Wir
kénnen daher die Ansicht nicht ganz anerkennen, die von einigen
Forschern, z. B. Agagobow, aufrecht erhalten wird, dass die
Zonulafasern Ableitungen von den dusseren Epithelzellen seien,
obgleich sie aus derselben Substanz wie die Intercellularsubstanz
zu bestehen schienen. Und dies auch trotz der Tatsache, dass,
wie ich schon friiher bemerkt habe, oftmals eine helle Proto-
plasmaverlangerung ausserer Zellen sich eine kurze Strecke weit
zwischen die inneren Epithelzellen einschiebt.

Diese Betrachtungen erklaren indessen das faserige Aus-
sehen der Intercellularsubstanz gewisser Regionen nicht, welches
von Schultze, Wolfrum, Lenhossék und auch von Mawas
bemerkt worden ist. Alle diese Forscher haben das faserige
Aussehen mit den Zonulafasern in Verbindung gebracht, indem
sie, ausser Lenhossék, annahmen, dass es von den Ver-
langerungen solcher Fasern herkomme, die in der gleichartigen
Intercellularsubstanz eingebettet sind. Sie haben jedoch nicht
erwihnt, ob dieses Aussehen auf die Region der Zonulaansatze
beschrankt war, oder ob es als charakteristisch bezeichnet werden
koénnte far alle Intercellularsubstanz durch das ganze Epithel,
sowohl zwischen den Zellen der iusseren, wie denen der inneren
Schicht. Darin aber liegt ein wichtiger Beweisgrund.

Bei meinen eigenen Untersuchungen habe ich bemerkt, dass
diese Faserung vor allem auf die Intercellularsubstanz begrenzt
ist, die zwischen jenen Zellen der inneren Epithelschicht lagen,
an welche sich Zonulafasern heften. Ich habe sie weder zwischen
den Zellen der Ausseren, noch zwischen denen der inneren Schicht
gefunden, die auf den Ciliarfortsitzen liegen, wo keine Zonula-
fasern entspringen. Zweitens erscheinen diese Faserchen zur
Zeit der Dickenzunahme dieser Substanz in den erwahnten

298 W.M. Baldwin:

Regionen. Drittens ist dieses Aussehen nur in dem Alter zu
finden, nachdem die Zonulafasern ihren Ansatz von den Apical-
fortsitzen der Epithelzellen in die Intercellularzwischenriume
verlegt haben. Endlich sind diese Faserchen in meinen Praparaten
immer in direktem Zusammenhang mit den Zonulafasern zu finden.

Hinsichtlich der Tatsache, dass neuere Forscher die Ansicht
mit Nachdruck betonen, dass fiir das richtige Studium der Zonula-
faserverlingerungen in der Intercellularsubstanz besondere Farbe-
methoden notig seien, z. b. die ,Heldsche Molybdansdure-Proto-
plasmafarbung“, muss ich nach meiner Erfahrung, — ich habe
die Bielschowskymethode angewandt — feststellen, dass die
bekannten Farbemittel wie Safranin oder Chloral - Himatoxylin
(Gage) vollstindig geniigen, um selbst die feinsten Faserchen zu
zeigen. Das einzige Erfordernis fiir ihre Behandlung besteht
darin, die Schnitte sehr stark zu tiberfarben und dann griindlich
zu wassern.

Wolfrums Ansicht, dass mehrere der Zonulafasern die
Zellen der inneren Epithelschicht durchzégen, ist nach der
Priifung von Tangentialschnitten des Epithels leicht als ungenau
zu erweisen, wie schon Mawas gezeigt hat. Bei meinen eigenen
Exemplaren habe ich sicherlich niemals bemerkt, dass eine Zonula-
faser eine Epithelzelle durchzog. Ich habe indessen die Zonula-
faserverlingerungen nicht durch die Intercellularsubstanz bis zur
lim. cil. ext. verfolgen kénnen. Wolfrum jedoch konnte sie bis
zu dieser Membran verfolgen, wo sie in kleinen runden An-
schwellungen endeten. Die letztere Bildung habe ich ebensowenig
auffinden kénnen.

Der zuletzt erwahnte Autor war imstande, bei seinen Sauge-
tierexemplaren Gliazellen zu finden, die von der Retina in der
Gegend der ora serrata in den Zonularaum wanderten, wo sie
faserihnliche Fortsitze ausschickten, die sich spater in Zonula-
fasern auflésten. Bei meinen eigenen Untersuchungen konnte
ich eine soleche Wanderung von Neurogliazellen nicht bemerken.
Jedoch habe ich aus Wolfrums Beschreibung dieser primitiven
Zellen geschlossen, dass die hellen, unregelmassigen, vielver-
zweigten Zellen, die ich von der ersten Stufe an beobachtete,
dieselben sind, die er als Neurogliazellen bezeichnet.

Im Jahre 1895 kam Rochon-Duvigneaud zu dem
Schlusse, dass die Zonulafasern ,une espéce particuliére de fibres

Die Entwicklung der Fasern der Zonula Zinnii. 299

conjonctives* waren. Spiter entdeckte Nussbaum die Bedeutung
solcher Zellen fiir die Entstehung fertiger Zonulafasern und er-
klarte sie fiir Bindegewebszellen und -fasern, die sich sekundar
mit der Linsenkapsel und dem Ciliarepithel verbinden. Meine
eigenen Schiliisse, die sich auf die Ergebnisse, welche ich in
dieser Arbeit niedergelegt habe, griinden, besonders aber der Mangel
eines Beweises in meinen Priparaten, die Arbeit Wolfrums
bestatigen zu kénnen, lassen mich eine Ansicht annehmen, ahnlich
der Nussbaums, dass namlich die Zonulafasern aus Mesen-
chymzellen hervorgehen und daher als Mesenchymfasern betrachtet
werden sollten.

Die Schliisse, zu denen ich gelangt bin, sind folgende:

I. Bei der weissen Maus haben die Zonulafasern sich aus
Mesenchymzellen entwickelt.

Il. Diese Fasern sind zuerst an die Apicalfortsitze der
Zellen der inneren Ciliarepithelschicht angeheftet.

Ill. Spater wechseln diese Zonulafasern ihren Ansatz und
dringen in die Intercellularsubstanz ein, die zwischen
den Zellen der inneren Ciliarepithelschicht liegt.

IV. Im fertigen Auge durchziehen die Zonulafasern die
Intercellularsubstanz nach der limitans ciliaris externa
hin; aber sie enden plotzlich, ehe sie dieses Gebilde er-
reichen.

V. Die Zonulafasern endigen nur an jenem Teile des Ciliar-
epithels, welches in den Talern zweier benachbarter
Ciliarfortsitze und zwischen den Ciliarfortsitzen und
der ora serrata liegt.

300

W. M. Baldwin:
Literaturverzeichnis.

Addario, C.: Sulla matrice del vitreo nell’ occhio umano et degli
animali. Riforma med. no. 17, 1901.

Derselbe: Sulla struttura del vitreo embryonale et dei neonati, sulla
matrice del vitreo et sull’ origine della Zonula. Pavia, p. 75, Tay. IX, 1902.
Derselbe: La matrice ciliare delle fibrille del vitreo, etc. Archivio di
Ottalmologia, p. 206, 1904.

Agababow, A.: Untersuchungen iiber die Natur der Zonula ciliaris.
Arch. f. mikr. Anat., Bd. L, p. 563—588, 1897.

Angelucci, Ant.: Uber Entwicklung und Bau des vorderen Uveal-

tractus der Vertebraten. Arch. f. mikr. Anat., Bd. XIX, 1881.

Derselbe: Physiologie générale de lVoeil, fonctions nutritives. Encyclopédie
franc. d’Ophtalmologie, t. II, p. 2—59, 1905.

Berger, E.: Bemerkungen zur Zonulafrage. Arch, f. Ophth., Bd. XXXI,
p. 3, 1886.

Derselbe: Anatomie normale et pathologique de Voeil. Masson, éd.
Paris 1893.

Derselbe: Historische Bemerkungen zur Anatomie der Ora serrata retinae.
Arch. f. Augenhlk., Bd. XXXII, p. 288, 1896.

Derselbe: Anatomie générale de Voeil, in Encycl. franc. d’Ophtalmol.,
t. I, p. 326—373, Paris, Doin, éd. 1903.

Claeys, G.: De la région ciliaire de la rétine et de la zonule de Zinn.
Bull. Acad. Roy. de Méd. de Belgique, 3 série, t. XX, p. 1301, 1886.
Derselbe: De la région ciliaire de la rétine et de la zonule de Zinn.
Arch. de Biologie, t. VIII, p. 623, 1885.

Cloquet, J.: Anatomie de Homme, t. II, p. 345—347, 1828.
Collins, E. J.: The glands of the ciliary body in the human eye. Trans.
Ophth. Soc., vol. XI, p. 53, 1890—1891.

Derselbe: The glands of the ciliary body, a reply to some recent
criticisms concerning them. Ophth. Review, vol. XV, 1896.

Czermak, W.: Zur Zonulafrage. Arch. f. Ophth., Bd. XXXI, 1, 1885.
Damianoff, G.: Recherches histologiques sur la cristalloide et sur
la zonule de Zinn. Thése de médecine, Montpellier 1900.
Dessauer, E.: Zur Zonulafrage. Klin. Monatsbl., p. 94, 1883.

vy. Ebner: In Kéllikers Handbuch der Gewebelehre des Menschen,
t. III, 2 partie, 1902. ;

Fischel: Die Regeneration der Linse. Anat. Hefte, Bd. XIV, 1900.
Derselbe: Weitere Mitteilungen iiber die Regeneration der Linse. Arch.
f. Entwicklungsmech., Bd. XV, p. 1, 1902.

Gage, S.H.: The Microscope, an introduction to microscopic methods
and to histology. 10th edit., Comstock Publ. Co., Ithaca, N. Y., 1908.
Haensell, P.: Recherches sur la structure et Vhistogénése du corps
vitré normal et pathologique. Thése de Paris 1888.
Iwanoff, H.: The vitreous humour. Strickers Handb., english ed.
Sydenham Society, p. 345—356, London 1873.

40.

41.

42.
45,

44.

45,
46.

Die Entwicklung der Fasern der Zonula Zinnii. 301

wih anoff, H. und J. Arnold: Mikrosk. Anat. des Uvealtractus und der

Linse. Graefe-Saemisch Handbuch, Erster Band, Cap. III, p. 265—-320.
Leipzig, W. Engelmann, 1874.

. Kessler, L.: Untersuchungen iiber die Entwicklung des Auges, an-

gestellt am Hiihnchen und Triton. Inaug.-Dissert., Dorpat 1871.

. Derselbe: Zur Entwicklung des Auges der Wirbeltiere. Leipzig, 1877.
. Kélliker, A.: Heidelberger Anatomenkongress. Anat. Anz., Ergiinz.-

Heft, p. 49, 1903.

. Derselbe: Die Bedeutung und Entwicklung des Glaskérpers. Zeitschr.

f. wissenschaft. Zoolog., LXXVI, I, 1904.

Leboucgq, G.: Contribution & l’étude de Vhistogénése de la rétine chez
les Mammiféres. Arch. d’anat. micros., pl. XVII und XIX, t. X, fase. III
und IV, p. 556—606, 1909.

. v. Lenhossék, M.: Die Entwicklung des Glaskérpers. Leipzig,

F.C. W. Vogel, 1903.
Derselbe: Die Entwicklung und Bedeutung der Zonulafasern, nach
Untersuchungen am Hiihnchen. Arch. f. mikr. Anat., Bd. 77, p. 280, 1911.

3. Lieberkiihn, N.: Uber das Auge des Wirbeltierembryos. Schriften

d. Gesellsch. z. Bef. d. ges. Naturwissensch. zu Marburg. Kassel, 1872.

. Derselbe: Beitrage zur Anatomie des embryonalen Auges. Arch. f.

Anat. und Entwicklungsgesch., 1879.

. Mawas, J.: Sur la structure de la rétine ciliaire. C.R. Acad. des

Sciences, 14 décembre, 1908.

. Derselbe: Recherches sur lorigine et la signification histologique des

fibres de la zonule de Zinn. C. R. de l’Assoc. des Anat., Réunion de
Marseille, avril 1908, p. 73—78.

. Derselbe: Note sur l’origine des fibres de la zonule de Zinn. C. R. Soc.

de Biol., 13 juin 1908, t. LXIII, p. 1029—1030.

. Derselbe: La structure de la rétine ciliaire et la sécrétion de V’humeur

aqueuse. C. R. Assoc. des Anat., 11 réunion a Nancy, avril 1909, p. 280—285.
Derselbe: La sécrétion de l’humeur aqueuse et la structure de la rétine
ciliaire 4 l'état normal et pathologique. Soc. d’ophthal. de Lyon,
1 Mars 1909, et Lyon méd., 1909.

Metzner, R.: Kurze Notiz iiber Beobachtungen an dem Ciliarkérper
und dem Strahlenbindchen des Tierauges. Verh. Naturf. Ges., Basel,
Bd. XVI, p. 481—492, 1903.

Nussbaum, M.: Entwicklungsgeschichte des menschlichen Auges.
Graefe-Saemisch, Handb. d. ges. Augenheilk., 2. Aufi., 1900.
Onfray: vidé Rochon-Duvigneaud et Onfray.

van Pée, P.: Recherches sur l’origine du corps vitré. Archives de
Biol., T. XIX, p. 317—385, 1903.

Rabl, C.: Uber den Bau und die Entwicklung der Linse. III. Teil.
Die Linse der Siiugetiere, Riickblick und Schluss. Zeitschr. f. wissensch.
Zool., Bd. LXVIII, p. 29, 1898.

Derselbe: Zeitschr. f. wissensch. Zool., Bd. LVI, 1899.

Derselbe: Zur Glaskérperfrage. Anat. Anz., Nr. 25, 1903.

302

17.

60.
61.

66.

W. M. Baldwin:

Retzius, G.: Uber den Bau des Glaskérpers und der Zonula Zinnii
in dem Auge des Menschen und einiger Tiere. Biol. Untersuch., neue
Folge, VI, no. 9, p. 67—87, pl. XXVUI—XXXII, 1894.
Rochon-Duyigneaud et Onfray: Expériences preparatoires & la
recherche des variations de concentration des liquides intra-oculaires et de
Jeur influence sur le tension de loeil. Soc. d’Opht. de Paris, 5 juillet 1904.
Salzmann, M.: Die Zonula ciliaris und ihr Verhaltnis zur Umgebung.
Eine anatomische Studie. Wien 1900.

Sbordone, A.: Sull’ origine delle fibre della zonula di Zinn. Ophthalmo-
logica, vol. I, fasc. I, p. 68—83, Table V, 1910.

Schoen, W.: Zonula und Ora serrata. Anat. Anz., p. 360, 1895.
Derselbe: L’occommodation dans l’oeil humain. Arch. d’ophtal., p.81, 1901.
Schultze, O.: Zur Entwicklungsgesch. des Gefaisystems im Siuge-
tierauge. Festschr. f. Kélliker, 1892.

Derselbe: Mikroskopische Anatomie der Linse und des Strahlenbindchens.
Graefe-Saemisch, Handbuch d. ges. Augenheilk., 2. Aufl., Leipzig 1900,
Lief. XVII.

Schwalbe: Lehrbuch der Anat. des Auges, 1887.

y. Spee, F. Graf: Uber den Bau der Zonulafasern und ihre Anordnung
im menschlichen Auge. Verh. der anat. Gesellsch., 16. Vers., Halle,
p. 236—241, 1902.

Vander Stricht, N.: L’histogénése des parties constituantes du
neuroepithelium acoustique, des taches et des crétes acoustiques et de
Vorgane de Corti. Arch. de Biologie, t. XXIII, fasc. IV, p. 541, 1906.
Van der Stricht, O.: Le neuro-épithélium olfactif et sa membrane
limitante interne. Mémoires de l’Ac. Roy. de Belgique, t. II, fasc. II,
p. 1—45, pl. Iu. Il, 1909.

Terrien, F.: Recherches sur la structure de la rétine ciliaire et
Vorigine des fibres de la zonule de Zinn. Thése médecine, Paris 1898.
Derselbe: La structure de la Rétina ciliaire. Arch. d’opht., Bd. X VIII, 1898.
Derselbe: Mode d’insertion des fibres zonulaires sur le cristallin et le
rapport de ces fibres entre elles. Arch. d’opht., t. XIX, p. 250-—257, 1899.
Topolanski: Uber Bau der Zonula und Umgebung nebst Bemerkungen
iiber das albinotische Auge. V. Graefes Arch. f. Ophth., Bd. XXXVII,
no. 1, p. 28—61, pl. I—II, 1891.

Toufesco, Sophie: Sur le cristallin normal et pathologique. Thése
medecine, Paris 1906, auch Annal. d’Oculist, Aoit, p. 101, 1906.

de Waele, H.: Recherches sur l’anatomie comparée de Voeil des
vertébrés. Journ. int. d’Anat. et Physiol., t. XIX, p. 1, auch Internation.
Monatsschr. f. Anat. u. Physiol., Bd. XIX, H.1 und 2, 1901.
Wolfrum, M.: Uber Ursprung und Ansatz der Zonulafasern im
menschlichen Auge. Graefes Archiv fiir Ophth., Bd. LXIX, no. 1,
p. 148—171, 1908.

Zinn, J. G.: Descriptio anatomica oculi humani iconibus illustrata
(Cap. IV. De humore vitreo, p. 23 et suiv.), apud videam Abrami
Vandenhoeck, Gottingae 1775.

Die Entwicklung der Fasern der Zonula Zinnii. 303

Erklarung der Abbildungen auf Tafel XIV und XV.

Fig. 12

Fig. 2.

Gegend der Zonula im Auge einer 12 Stunden alten weissen Maus.
A = distale Partie der Linse; B = ihre Kapsel; C = ein Stiick
Retina, innen begrenzt von der Mm. limitans interna. Vom Ciliar-
epithel ist nur der proximale Teil abgebildet, der in Beziehung zur
Entwicklung der Zonulafasern (D) steht; E und F = Blutgefisse;
G = Stiitzmembran eines Blutgefisses aus Fortsaitzen einer Mesen-
chymzelle gebildet; den stark gefirbten Zellfortsitzen liegen
Kérnchen auf. Einige dieser Fortsitze kénnen bis zu den apicalen
Fortsitzen der inneren Lage der Ciliarepithelien (H), verfolgt
werden. J — grosse, unregelmiissig gestaltete und helle Mesen-
chymzelle mit zahlreichen feinen, hellen Fortsitzen. Dieser Zellen-
typus ist auf die Gegend der Zonula beschrankt und kommt im
Glaskérper nicht vor. Aus solchen Zellen entspringen die Zonula-
fasern des erwachsenen Tieres. Bei der 12 Stunden alten Maus
erreichen, wie Fig. 1 zeigt, die Fortsatze dieser Zellen die Linse
noch nicht. Ihre zahlreichen Fibrillen bilden ein dichtes Netzwerk
auf dem proximalen Abschnitt des Ciliarepithelium, woran sich die
Fibrillen schliesslich festheften. An jedem zugespitzten Fortsatz
einer Epithelzelle sitzt eine Fibrille. Diejenigen epithelialen Zellen,
welche distal zu dem von den Fortsatzen der hellen Mesenchymzellen
gebildeten Netzwerk liegen und spiaterhin die Ciliarfortsaitze decken,
verlieren ihre apicalen Fortsitze und sind demgemiss beim Er-
wachsenen mit der Zonula nicht mehr verbunden. Die Epithelien
der ora serrata (K) dagegen und die nachsten distalen liegen im
Gebiet der Zonula des Erwachsenen. Es gelang mir festzustellen,
dass bei der 12 Stunden alten Maus im Bereich der Zonula die
Hauptmasse des fibrillaren Netzwerks aus den Fortsitzen der hellen
Zellen und nicht von solchen der Epithelzellen gebildet wird. Hervor-
gehoben zu werden verdient auch, dass die Intercellularsubstanz der
Epithelien an der Zonula nicht dicker ist als distal davon, und dass
um diese Zeit keine Faser des Netzwerks mit der Intercellular-
substanz der Epithelien verbunden ist. Vergr. 500.

Aus mehreren Schnitten zusammengesetzte Ansicht der Zonulagegend
einer 10 Tage alten weissen Maus. A = distales und B — proxi-
males Epithel eines Teiles der Linse mit ihrer Kapsel C. D =
Membrana hyaloidea als eine diinne Membran die Zonulagegend yom
Glaskérper trennend und von der proximalen Partie der Linse bis
zur ora serrata sich erstreckend (KE). Die Retina (F) hat mehrere
Zellenlagen und ist innen von der Membr. limitans interna (G) be-
grenzt. H = Stiibchen und Zapten, wohl entwickelt. Die Membr.
limitans externa der Retina (J) ist distal bis zwischen die beiden
Epithellagen des Ciliarkérpers zu verfolgen, wo sie zur Membr.
limitans ciliaris externa (K) wird. Die Membr. limitans interna
retinae hingt mit der Intercellularsubstanz der Epithelien an der

504

W. M. Baldwin:

ora serrata zusammen, direkt proximal von der Anheftung der
Membr. hyaloidea. L = ein Stiick Iris, M = ein Ciliarfortsatz.
Vom Wpithel der Ciliarfortsitze entspringen keine Zonulafasern;
solche (N), die mit dem Epithel der Zonulagegend zusammenhiingen,
streichen iiber die freie Fliiche des Epithels der Fortsatze hin. Die
Zonulafasern sind halbschematisch eingezeichnet und finden sich am
reichlichsten proximal vom Linsenaiquator (O). In der Zonulagegend
sind zwei Blutgefasse getroffen; das eine enthalt im Inneren ver-
schiedene Blutkérperchen und eine Mesenchymzelle (P) auf seiner
Wand. Die Fortsatze dieser Mesenchymzelle sind bis an das Ciliar-
epithel zu verfolgen; viele Zonulafasern gehen in die Intercellular-
substanz des Epithels, distal haben noch verschiedene Epithelzellen
zapfenartige Fortsitze. Vergr. 250.

Nach einem Priiparat von einer 14 Tage alten weissen Maus mit
offener Lidspalte genau kopiert. A = Retina, B = Ciliarepithel.
(Die Retina ist im Schnitt nach vorn verschoben.) C = ora serrata;
D = Membr. limitans interna; EK = Membrana hyaloidea; die beiden
Membranen zerfallen in mehrere Lamellen; jede derselben hingt
mit der Intercellularsubstanz der inneren Lage der Ciliarepithelien
zusammen. Die Intercellularsubstanz ist an dieser Stelle dicker
als sonstwo in der Zonulagegend. F = Membrana limitans externa
retinae an der ora serrata verdickt;: G == Membrana limitans
externa ciliaris mit der vorigen zusammenhiingend und die beiden
Epithellagen trennend. Hine achte Membrana limitans interna ciliaris
ist an diesem Priparat nicht nachweisbar. Das Epithel der iusseren
Lage ist deutlich in Form, Grésse und Granulierung von dem der
inneren Lage verschieden. H = Pigmentzellen der Retina. J =
Teil eines Ciliarfortsatzes; auf seinem Epithel liegt eine der grossen
Mesenchymzellen, wie sie um diese Zeit in grésserer Zahl sich finden.
Die Zelle (K) hat zwei Fortsitze; der eine zieht zur Linse und ist
kurz abgeschnitten, der andere zerfasert sich auf der Oberfliche
des Ciliarfortsatzes. Eine andere grosse Mesenchymzelle (L) liegt
im Zonulabezirk auf der Membrana hyaloidea; der eine ihrer ver-
zweigten Fortsitze endet auf der Oberfliiche des Epithels der ora
serrata. Zwischen diesen beiden Zellen liegen die Zonulafasern ;
viele derselben gehen in diesem Stadium zwischen die Epithelzellen.
Im Vergleich zu dem Stadium yon 12 Stunden ist zu bemerken,
dass bei dem 14 Tage alten Tier die Zahl der in eine kurze Spitze
ausgezogenen Epithelzellen bedeutend abgenommen hat. Zuweilen
sieht es aus, als wenn einige Zonulafasern in der Nahe der ora
serrata durch Epithelzellen bis zur Membrana limitans externa
reichten. Untersucht man solche Stellen aber sorgfaltig genug, so
stellt sich heraus: dass alle diese Zonulafasern auf der dem Be-
obachter zugewandten Seite der Zellen in die Intercellularsubstanz
eingebettet sind. Keine Zonulafaser geht durch eine Zelle und keine
reicht bis an die Membrana limitans externa ciliaris. Vergr. 500.

Fig. 4.

Die Entwicklung der Fasern der Zonula Zinnii. 305

Von einer 27 Tage alten weissen Maus. Ciliarepithel zwischen ora
serrata (A) und dusserem Rand der Iris (B). C = der mesenchymatische
Kern eines Ciliarfortsatzes mit Blutgefiiss. Die musivischen Epithel-
zellen (D) deuten an, dass der Ciliarfortsatz etwas schrag getroffen
ist. E — verzweigte Fortsaitze tiefer gelegener Mesenchymzellen,
die in die Intercellularsubstanz der fusseren ciliaren Epithellage
iibergehen. An keiner Stelle kann ein Ubergang dieser Fasern, in
Zonulafasern nachgewiesen werden. Die Abbildung zeigt deutlich,
dass die Intercellularsubstanz im inneren Zellenlager des Ciliar-
fortsatzes nicht verdickt ist, im scharfen Gegensatz zum eigentlichen
Zonulagebiet nahe der ora serrata. Man findet am Innenrande der
Epithelien keine Spitzen mehr wie friiher; alle Zonulafasern heften
sich an die epitheliale Intercellularsubstanz an. Diejenigen Fasern,
welche scheinbar vom Ciliarkérper selbst entspringen, kénnen proximal
iiber den darunter gelegenen epithelialen Rand nach dem Zonula-
gebiet an der ora serrata verfolgt werden. Diese Fasern erscheinen
unter der Form einer Membrana limitans ciliaris interna. Vergr. 500.

i AB +4 ieee

Wei

r Pe ats he. Ooh Saar ‘ he

Archiv f mikroskon. Anatomie Bd.LXXX, Abt.

|e a pees es

K

1

Tat XV.

Archiv fmikroskop. Anatomie Ba. IXXX, Abt.

[ Reprinted from Scimnce, N. S., Vol. XXXVI., No. 916, Pages 90-92, July 19, 1912)

RHYTHMICAL ACTIVITY OF ISOLATED HEART MUSCLE CELLS IN
VITRO

In previous communications!:? I pointed
out that the heart muscle of chick embryos
will beat rhythmically for many days when
suspended in the media of a tissue culture
and from such transplanted tissue there is an
active growth of cells into the surrounding
media. Braus? has repeated these experi-
ments, using the hearts of embryo frogs and
toads and he has found that these isolated
beating hearts react to electrical and chem-
ical stimuli similar to the intact heart.
Braus also noted that the cells which grew
from the hearts of cold-blooded animals were
living at the end of three months. Very
recently, Carrel* by the use of the method of
repeated transplantation of the tissue from a
culture to a fresh medium (Carrel and Bur-
rows) has attempted to prolong the life and
function of heart muscle in vitro. His ex-
periments show that the rhythm which I
noted in fragments of embryonic chick hearts
ean be prolonged, although intermittently,
for a period of 85 days. The results of these
experiments substantiate, therefore, the former
well-known fact, namely, that strips of heart
muscle, both of cold and warm blooded ani-
mals (Erlanger), will beat for some time when
placed in the proper media. In none of these
eases could one rule out, however, the possi-
bility of the existence of nerve ganglia or
some possible precursor in the young embry-

* Burrows, M. T., 1911, Jour. Exp. Zool., Vol.
10, 63.

? Burrows, M. T., 1912, Anat. Record, Vol. 6,
141,

*Braus, H., 1912, Weiner Med. Wochschr., No.
44,

“Carrel, A., 1912, Jour. Exp. Med., Vol. XV.,
516.

onic hearts, which might initiate rhythmical
contractions.

During the present year experiments have
been made to determine the conditions which
would prolong the life and allow the develop-
ment of functional activity in the cells which
had grown and differentiated in the culture.
These experiments have shown that the newly
grown, cellular syncytia and the isolated single
heart muscle cell can become functionally ac-
tive, beating with a rhythm similar to that of
the intact heart.

Pieces of the hearts of chick-embryos of all
ages and of young hatched chickens were
used. A growth of tissue, composed almost
entirely of muscle cells, occurred from all
pieces when suspended in the media of both
types of cultures, (1) the ordinary hanging
drop culture (the plasma modification!) of
the method of Harrison® and (2) a large modi-
fied type of culture. This apparatus is so ar-
ranged as to supply the tissues continuously
with fresh media and to wash away the waste
products without in any way disturbing the
growing cells. I described this method in de-
tail before the American Association of
Anatomists, December 27, 1911.2 Serum was
used as the fluid medium in the latter type of
culture.

Rhythmical activity of the newly grown
cells was noted in 3 out of 15 of the large
type of cultures (No. 2), and in 2 out of 150
of the ordinary hanging drop cultures. These
cells were located definitely within the clot
and had a clear cytoplasm which contained
very few fat droplets. The rhythmical activ-

* Harrison, R. G., 1907, Proc. Soc. Exp. Biol.
and Med., 140; 1910, Jour. Exp. Zool., Vol. 9,
787.

y) | §CLENCE

ity did not occur during the active outwander-
ing of the cells but, later, after they became
permanently located in a definite portion of
the clot and were undergoing slow multipli-
cation and differentiation. In one culture

rhythm occurred as early as the fifth day,’

while in others as late as the fourteenth day
of the life of the culture. The greater num-
ber of positive results in the large type of
culture (No. 2) can be associated with the ac-
tive and continuous growth of the tissue
over a sufficient period of time. Active
erowth. and a regular rhythm has been ob-
served in these cultures for 80 days, while in

the hanging drop eulture the active growth.
and the regular rhythm. cease after the third,
The growth then’ becomes .
gradually less and the rhythm intermittent, |

or fourth day.

ceasing entirely after 10 or 18 days unless the
tissue is transferred to a new medium. The
method of repeated transplantation from the
culture to a new medium has not as yet been
sufficiently developed to allow any increase
in the life and the activity of the newly grown
cells. At each transfer of the tissue the ac-
tively growing and multiplying cells are de-
stroyed and a new growth takes place from
those more latently active cells in or about the
tissue mass.

The original pieces of heart muscle trans-
planted to a tissue culture vary as to their

rhythmical activity in relation to the portion
of the heart from which they are taken as well

as the age of the embryo. Pieces of the
auricle, especially of that part situated near
the entrance of the veins, taken from embryos
of all ages and from young hatched chickens,
beat when suspended in plasma. The pieces
of the ventricle do not beat when taken from

embryos older than 10 days, uriless special
methods of preparation and treatment are:
used.

Rhythmically beating cells have been grown
from the contracting pieces of the hearts of
young embryos and from one piece of the ven-
tricle of a fourteen-day chick embryo. The
absence of movement in the original mass of ,
tissue of this culture facilitated greatly the’
study of the delicate contractions:of the newly
grown cells. The syncytial network which
surrounded the original tissue and one isolated
cell were beating rhythmically. This cell was
situated far out in the clear medium away from

all other tissues and beat with a rhythm inde-

pendent in phase from that of the syncytium.
The rate of all beating cells in this culture was.

the same, 50 to 120 per minute, or a* rhythm
‘typical for rhythmical beating; re of ven-

tricular muscle.

The experiments show: (1) that 4 cells
which have grown and differentiated sin a tis-
sue culture can later assume their character-
istic function; (2) that rhythmical contrac-
tion similar to that observed in the embryonic
heart can occur in an isolated and single heart
muscle cell; (8) that the rhythmically con-
tracting cells can be grown not only from the
pieces of hearts of young embryos, but from
the heart muscle of a fonts er chick
embryo.

These experiments, therefore, give direct
evidence for the myogenic theory of the heart
beat.

Montrose T. Burrows, M.D.

ANATOMICAL LABORATORY,
CoRNELL UNIVERSITY MEDICAL COLLEGH,
* NEw York City

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Aus dem anatomischen Laboratorium des Cornell University
Medical College, New York City (Vorstand: Prof. Dr. Ch. R.
Stockard).

Rhythmische Kontraktionen der isolierten Herzmuskel-
zelle ausserhalb des Organismus.

Von Dr. Montrose T. Burrows.

Durch friihere Untersuchungen‘) ist bewiesen, dass der
Herzmuskel des Hiihnerembryos, auf geeignete Nahrbéden
implantiert, sich wahrend 8 Tagen rhythmisch bewesgt. Dieser
Befund wurde von Braus2) (Unken- und Froschembryonen)
und von Carrel’) (Hiihnerembryonen) bestatigt. Aus
diesen Untersuchungen geht deutlich hervor, dass die funktio-
nelle Tatigkeit des Gewebes ausserhalb des Organismus lange
Zeit erhalten bleiben kann. FEigentiimlich bei den Ge-
webekulturen*) ist die Auswanderung der Zellen des ur- .
spriinglichen Gewebestiickes in den umgebenden Nahrboden
hinein, welcher Vorgang dem eigentlichen Wachstum voraus-
geht. Weitere Aufgabe ware nun zu erforschen, ob die
Funktionen solcher ausgewanderten, isoliert liegenden Zellen
nach event. Teilung und Differenzierung denen des Mutter-
gewebes gleichwertig sind. Bei der vorliegenden Unter-
suchung hat es sich tatsachlich herausgestellt, dass die iso -
lierten Herzmuskelzellen rhythmische Bewegungen aus-

1) M. T. Burrows: Compt. rend. soc. de biol. 1910, LX. 290
Jour. Exper. Zool. 1911, X, 63.

2) H. Braus: diese Wochenschrift 1911, S. 2421 u. 2237; Wiener
med. Wochenschr. 1911, No. 44.

3) A. Carrel, Jour. Exper. Med. 1912, XV, 516.

4) Der Ausdruck ,,Gewebekultur“ wird hier in Analogie
mit Bakterienkultur gebraucht. Das Prinzip der Gewebekultur
besteht darin, dass man ein steril entnommenes Gewebestiick
auf ebenfalls sterilem Nihrboden derart implantiert bezw. sus-
pendiert, dass die Wachstumserscheinungen von Zeit zu Zeit
bei Brutofentemperatur verfolgt werden k6nnen.

1

a ae

fiihren. Bei der allgemeinen Bedeutung welche diesem Re-
sultate zukommen diirfte, soll die Methodik, sowie ihre ge-
schichtliche Entwicklung eingehend beschrieben werden.
Denn es ist im hdchsten Grade wahrscheinlich, dass es
auch gelingen wird, bei Anwendung geeigneter Methodik die
mehr spezifischen Funktionen anderer hoch differenzierten
Gewebé in vitro naher zu verfolgen.

Die erste praktische Anwendung der Gewebekulturmethode,
wobei Gewebe lingere Zeit ausserhalb des Organismus ge-
halten und ihre weitere Entwicklung gleichzeitig kontinuierlich
beobachtet werden konnte, wurde von Harrison bei seinen
Untersuchungen iiber Nervenfaserentwicklung gemacht. An friiheren
Untersuchungen iiber das Ueberleben der Gewebe und Organe
hat es freilich nicht gefehlt, aber solche sind meistens von
ganz anderen Gesichtspunkten aus unternommen worden. Die
Versuche von Wentcher®), Liunggren’), Carrel’) wu. a.
haben ergeben, dass es moglich ist, tierische Gewebe lange
Zeit ausserhalb des Organismus am Leben zu erhalten. Bei
den Untersuchungen von Ranvier’), Jolly’), Beebe und
Ewing’) u. a. wurde hauptsadchlich auf bestimmte Aeusse-
rungen funktioneller Tatigkeit des in vitro lebenden Ge-
webe geachtet. Im Jahre 1897 gab Leo Loeb”) an, dass es ihm
gelungen sei, das Wachstum des Gewebes im geronnenen Blutserum
oder Agar ausserhalb des Organismus zu verfolgen. Ndaheres iiber
die Technik und Resultate gibt er nicht an. Derselbe Autor (1902)
beschrieb Versuche iiber das Wachstum der Haut. Diesmal nahm
er Blécke geronnenen Blutserums resp. Agar, spaltete dieselben,
fiihrte das Gewebestiickchen dort ein und brachte das Ganze in das
Unterhautzellzewebe des Tieres. Weiter untersuchte er bei der
Wundheilung, wie das Epithel in den Schorf hineinwuchs.

Die erste Arbeit Harrisons*) auf diesem Gebiet stammt aus
dem Jahre 1907. Die Methode bestand darin, dass Gewebestiicke
junger Froschembryonen in einem hangenden Tropfen Froschlymphe
suspendiert wurden. Kurze Zeit nach dem Einbringen des Gewebe-
stiickes gerann natiirlich die Lymphe und bot auf diese Weise ein
festes Substrat fiir das weitere Wachstum des Gewebes. Mit dieser

>) J. Wentcher: Berl. klin. Wochenschr. 1894, 979; Zieglers
Beitr., XXIV.

6) Liunggren: D. Zeitschr. f. Chir. 1898, XLVII, 609.

*) A. Carrel: Jour. Exper. Med. 1910, XII, 460.

8) Ranvier: Traité Technique d’Histologie, Paris 1889.

®) Jolly: Compt. rend. soc. de biol. 1903, LV, 1266.

1) Beebe und Ewing: British Med. Jour. 1906, II, 1559.

4) Leo Loeb: Ueber die Entstehung von Bindegewebe, Leuko-
zyten und roten Blutkérperchen aus Epithel und iiber eine Methode.
isolierte Gewebsteile zu ziichten: Chicago 1897, 41. Archiv f. Ent-
wicklungsmechanik d. Organ. 1902, XIII, 487.

*) R. G. Harrison: Proc. Soc. Exper. Biol. and Med. 1907,
IV, 140; Anat. Record 1908, II, 385; Harvey Lectures, Philadelphia,
1907—1908; Jour. Exper. Zool. 1910, IX, 787.

aie BT Le

Methode hat Harrison das Wachstum des embryonalen
Zentralnervensystems, des Muskels und der Haut in vitro
auf schlagende Weise nachgewiesen. Bei diesen Versuchen
wanderten die Zellen langs der Fibrinfiden aus dem _ ur-
spriinglichen Gewebestiick heraus, um sich dort im Fibrin- ~
geriist weiter zu _ differenzieren. Aus dem Neuroblastenproto-
plasma entwickelte sich selbsttatig der Achsenzylinder. Dadurch
wurde die Richtigkeit der Hisschen Annahme von Harrison ein-
deutig bewiesen. Gleichzeitig hat Harrison die Grundlage fiir
weitere Versuche iiber das Wachstum des Gewebes in vitro ge-
schaffen.

Unter Anwendung der von Harrison angegebenen Prinzipien
habe ich**) im Laboratorium Harrisons die Methode dadurch
modifiziert und vereinfacht, dass Blutplasma statt Lymphe zur
Verwendung kam. Die Anwendung von Plasma ist deswegen ein
wesentlicher Fortschritt, weil.man auf diese Weise geeignete Nahr-
béden fiir das Gewebe verschiedener Tiere leicht gewinnen kann.
Dies ist die zurzeit am meisten gebrauchte Methode der Gewebe-
kultur. Neuerdings ist sie von Carrel und mir im Handbuch der
biochemischen Arbeitsmethoden Bd. 5, Teil 2, S. 838) ausfiihrlich
wiedergegeben worden. Ich untersuchte damals das Wachstum em-
bryonalen Gewebes vom Frosch und Huhn im Frosch- resp. Hiihner-
plasma und konnte die Resultate Harrisons bestiatigen. Ich
konnte ferner Kernteilungsfiguren haufig nachweisen. Noch wichtiger
erschien mir die Tatsache, dass die funktionelle Tatigkeit des
Gewebes in solchen Praparaten lange Zeit erhalten blieb. Beispiels-
weise fiihrte das Herz von 60 Stunden alten Hiihnerembryonen rhyth-
mische Kontraktionen bis zum achten Tage nach der Herstellung des
Pradparates aus. Wegen der prinzipiellen Bedeutung dieser Tat-
sachen haben Carrel und ich**) mit Hilfe der Plasmamethode den
naheliegenden Gedanken verfolgt und das Wachstum der Organ- und
Gewebestiicke verschiedener Tiere sowie deren Embryonen unter-
sucht. Bei diesen Versuchen gelang zum ersten Male die Kultivierung
von Organen und Gewebestiicken erwachsener Tiere. Das Wachstum
zahlreicher bésartiger Geschwiilste (auch von Menschen) in vitro
wurde gleichfalls studiert. Kurz darnach haben Lambert und
Hanes*) ahnliche Versuche mit den Mause- und Rattentumoren
angestellt. Dabei haben sie die wichtige Beobachtung gemacht,
dass diese Geschwulst- und Gewebestiicke auch in artiremdem.
Plasma wachsen. M. R. und W. H. Le wis’**) haben das Wachstum
embryonalen Hiihnergewebes in verschiedenen fliissigen und festen
Nahrbéden (Agar) untersucht. Sie konnten als erste das Wachstum
dieses Gewebes in einfachen Salzlésungen konstatieren. In seiner
ersten Mitteilung hat Harrison auf die Notwendigkeit eines festen

13) M.T.Burrows: loc. cit.; Journ. Am. Med. Ass. 1910, LV, 2057.

4) Carrel und Burrows: Journ. Am. Med. Assn. 1910, LV,
1379, 1554, 1732; Compt. rend. soc. de biol. 1910, LXIX, 293, 298, 299,
332; Jour. Exper. Med. 1911, XIII, 387, 571.

1%) Lambert und Hanes: Journ. Am. Med. Assn. 1911, LVI,
33, 791; Jour. Exper. Med. 1911, XIII, 495; XIV, 129, 453.

16) M. R. und W. H. Lewis: Johns Hopkins Hospital Bull. 1911,
XXII, 241; Anat. Record 1911, V, 277; VI, 207.

eae” Nae

Substrats, z. B. Fibringeriist, geronnener Lymphe fiir die Ent-
wicklung von.Nervenfasern und Zellen schon hingewiesen. Als Be-
stiitigung dieser Anschauung konnte ich*’) Form und Gestalts-
st6rungen, wie z. B. Abrundungen und Verkleinerungen von Spindel-
zellen, die von Fibrinfiiden losgelockert wurden, beobachten. Wenn
solche Zellen wieder in Beriihrung mit einem festen Korper kommen,
strecken sie sich alsdann zu einer langlichen oder unregelmassigen
Gestalt aus. Dieser Befund wurde spater von Carrel und mir an
Tumorzellen bestatigt. Welche Gestalt schliesslich angenommen
wird, ist von der Art des festen Substrates abhingig. Harri-
son?) hat nachtriiglich gezeigt, dass die Zellen, welche sich bei An-
wendung eines fliissigen Nahrbodens, wie bei den Versuchen von
M. R. und W. H. Lewis, an dem Wachstum beteiligen, sich dem Deck-
elas stets anfiigen. Es konnte ferner gezeigt werden, dass Spinngewebe
(Harrison), baumwollene resp. seidene Faden*) geniigende Stiitze
fiir ein gerichtetes Wachstum in fliissigen Nahrb6den darbieten.

Carrel und ich™) haben auf die Mdéglichkeit sukzessiver
Transplantationen von Gewebezellen aus Kulturen auf frischen Nahr-
boden hingewiesen; sekundére und tertiare Kulturen erhielten wir
leicht. Gelegentlich ihrer Versuche iiber Krebsimmunitaét haben
Lambert und Hanes”) von diesem Prinzip Anwendung gemacht
und konnten dabei die Zellen der Subkulturen geniigend lang am
Leben erhalten, um die Wachstumserscheinungen der Zellen des
urspriinglichen Gewebes in einer aufeinanderfolgenden Reihe von Sub-
kulturen auf Plasma verschiedener Herkunft zu untersuchen. Mittelst
sukzessiver Transplantation hat Carrel”) nachher Gewebe lange
Zeit hindurch am Leben erhalten k6nnen.

Gerade in der letzten Zeit ist die Plasmamethode auf die
verschiedenen biologischen Probleme vielfach angewendet
worden. Hier sei besonders auf die Arbeiten von Ruth”),
Lambert”), Carrel und Ingebrigtsen”), Loeb

17) M: T. Burrows; locxcit

18) R.G. Harrison: Science 1911, XXXIV; Anat. Record 1912,
ViEVisic

%) Carrelund Burrows: Jour. Exper. Med. 1911, XIV, 244.

*) Carrel und Burrows: Compt. rend. soc. de biol. 1910,
LXIX, 329, 365; Jour. Exper. Med. 1911, XIII, 416.

4) Lambert und Hanes: Jour. Am. Med. Assn. 1911, LVI,
587; Jour. Exper. Med. XIII, 505.
*) Carrel: Jour. Am. Med. Assn. 1911, LVII, 1611; und 1912,
OG, (Clt-
_ %) E. S. Ruth: Cicatrization of Wounds in vitro. Jour.
Exper. Med. 1911, XIII, 422.

_ *) R. A. Lambert: The Production of Foreign Body Giant
Cells in vitro. Jour. Exper. Med. 1912, XV, 510.

*) Carrel und Ingebrigtsen: The Production of Anti-

bodies by Tissues living outside the Organismus. Jour. Exper. Med.
1912, XV, 287. — Carrel: Berl. klin. Wochenschr. 1912, No. 12;
Jour. Exper. Med. 1912. ‘

i ae

— 5 —

md Fleisher”); Mada”) Oppel), Braus”) und
W eil®) hingewiesen. Es sei bei dieser Gelegenheit darauf
aufmerksam gemacht, dass in den Arbeiten von Hada und
von Oppel die Plasmamethode zur Kultivierung der Gewebe’
wiederholt als die ,C arrelsche Methode“ bezeichnet wird.
Aus dem Vorhergehenden ist aber ersichtlich, dass die wesent-
lichsten Fortschritte auf dem Gebiete des Gewebezellen-
lebens ausserhalb des Organismus fast ausschliesslich mit der
Plasmamethode gewonnen worden sind. Auch ist die Plasma-
methode nur eine Modifikation der Harrisonschen Me-
thode, und die Modifikation, namentlich die Anwendung von
Blutplasma statt Lymphe, ist von mir im Laboratorium
Harrisons ausgearbeitet worden. Somit war Carrel
auf keine Weise an der Entwicklung dieser Methodik be-
teiligt.

Methodik: Es wurden Praparate nach zwei Methoden
angefertigt: 1. nach der Methode des hangenden Plasma-
tropfens (Harrison, Burrow s), 2. nach einer von mir
kiirzlich ausgearbeiteten Methode, bei der das Gewebe fort-
wahrend mit frischem Serum umspiilt wird. Beim zweiten
Verfahren wird eine bessere Annaherung an die Verhiltnisse im
lebenden Organismus erzielt, indem dafiir gesorgt wird, dass
die Nahrungsstoffe kontinuierlich erneuert werden, wahrend
die Bestandteile des zellularen Stoffwechsels gleichzeitig ent-
-fernt werden. Denn es ist ohne weiteres klar, dass selbst bei
Anwendung des geeignetsten Nahrbodens eine Aenderung der
Wachstumsgeschwindigkeit auftritt, wenn eine Anhaufung der
Bestandteile des zellularen Stoffwechsels stattgefunden hat.
Mit Riicksicht auf diese Faktoren habe ich diese Methode er-
sonnen, in der Hoffnung, dass man dadurch die Dauer des
Wachstums verlangern kann, um Aufschluss iiber den Stoff-
wechsel sowie andere Aeusserungen funktioneller Tatigkeit
zu gewinnen.

2) Loeb und Fleisher: Ueber die Bedeutung des Sauer-
stoffs fiir das Wachstum der Gewebe von Saugetieren. Bioch. Zeit-
schrift 1911, XXXVI, 98.

27) S Hada: Die Kultur lebender Korperzellen. Berl. klin.
Wochenschr. 1912, No. 1, 11.

28) A. Oppel: Ueber die Kultur von Sdugetiergewebe ausser-
halb des Organismus. Anatomischer Anzeiger 1912, XL, 464. Archiv
i. Entwicklungsmechanik d. Organ. 1912, XXXIV, 132.

2) Braus: loc. cit.

30) Weil: Some Observations on the Cultivation of Tissues in
vitro. The Jour. Med. Research 1912, XX VE:

=, SB a

Es sei hier an das wesentlichste der Methodik erinnert *4) (vergl.
hierzu Fig. 1). Mittelst eines Dochtes wird das Medium von einem
Behalter (a) durch die Kulturkammer (b) geleitet, um dann in einen
anderen Behiilter (c) aufgenommen zu werden. In der Kulturkammer
wird der Docht in seine einzelnen Fasern, welche sich an der Ober-
fliche des Deckglischens festsetzen und ein Kapillarnetz
bilden, zerzupft. Das Gewebe wird in kleine Stiicke zerschnitten
und in das offene Netz von Baumwollfasern gelegt und hier durch
das Gerinnen der hinzugefiigten Plasmatropien festgehalten. Der
fliissige Nahrboden bewegt sich langsam am Docht entlang durch die
Kultur und sammelt sich in der Aufnahmekammer (c). Die Nahr-
fliissigkeit an den Geweben wird auf diese Weise fortwahrend ver-
andert, ohne dass die wachsenden Zellen in irgend einer Weise ge-
stort werden.

Fig. 1. Kulturapparat.
% der natiirlichen Grdsse.

Die Zufuhrkammer (a) ist aus Glas gemacht und besitzt zwei
Abteilungen. Die Lésung wird von dem horizontalen Behalter nach
oben in die vertikalen Abteilungen am oberen Ende des Dochtes ge-
fiihrt. Hierdurch wird eine genaue Regulierung der pro Zeiteinheit
benutzten Menge erméglicht. Serum wurde als fliissiger Nahrboden
in dieser Reihe von Experimenten benutzt.

Herzen von Hiihnerembryonen zu allen Zeiten des em-
bryonalen Lebens, sowie auch die von ausgebriiteten Hiihn-
chen wurden zu den Kulturversuchen verwendet. Von den
jungen Embryonen wurde meist das ganze Herz, von den
alteren sowie 6fter auch von den jungen nur ein heraus-

a) Vorgetragen in der Sitzung der American Association of Ana-
tomists am 27. Dezember 1911. Anatomical Record, VI, 141.

SR ak

geschnittenes Stiickchen Herzmuskel auf den Ndahrboden
gebracht. :

Das ganze Herz der 60—96stiindigen Embryonen
schlagt rhythmisch in Kultur mit einer Frequenz von 50 bis
120 Schlagen in der Minute. 3

Ob die herausgeschnittenen Stiicke pulsieren oder nicht,
ist sowohl vom betreffenden Teile des Herzens, welchem sie
entnommen werden, als auch vom Alter des Embryo ab-
haingig. Ventrikelstiicke von 60 stiindigen bis 10 tagigen Em-
bryonen, Stiicke des Vorhofs, insbesondere aus der Nahe der
Venen der Embryonen beliebigen Alters und des jungen Hiihn-
chens zeigten rhythmische Kontraktionen. Die Ventrikel-
stiicke von dlteren Embryonen haben nicht rhythmisch pul-
siert **). Die Frequenz des Rhythmus ist beim Vorhof grosser
als beim Ventrikel; im ersten Falle betragt sie 150 bis 220,
wahrend im zweiten Falle nur 50 bis 150 Schlage in der Minute.
Der Rhythmus bei den gewohnlichen Hangetropfenkulturen
bleibt bis zum 3. oder 4. Tage regelmassig und wird spater
unregelmdssig. Dieser intermittierende Rhythmus kann in
solchen Praparaten bis zum 17. Tage dauern. In den gr6ésseren
Kulturen mit bestaéndiger Zu- und Abfuhr frischen Serums
bleibt der Rhythmus regelmdssig, sogar bis zum 30. Tage.

Wachstum. Die Wachstumserscheinungen lassen sich
in zwei Perioden zerlegen, erstens die der lebhaften Aus-
wanderung der Zellen des urspriinglichen Gewebestiickes in
den es umgebenden Nahrboden hinein; zweitens die der Tei-
lung und Differenzierung. Die erste Periode faingt gegen Ende
des ersten Tages an und dauert vom 5. Tage bis in die
2. Woche bei beiden Arten der Kulturen. Sie ist sowohl
durch die Bildung eines synzytiumahnlichenFilzwerkes um das
Gewebe herum als auch durch die Auswanderung einer
grossen Anzahl Herzmuskelzellen (Fig. 2) gekennzeichnet. In
_ allen Praparaten wurde die Auswanderung von Zellen, welche
nach genauer Untersuchung als Herzmuskelzellen identifiziert
werden konnten, beobachtet. Der Umfang und die Dauer des
Wachstums ist von der Festigkeit der Schichtdicke des Nahr-
bodens abhangig. Wdahrend der zweiten Periode erfolgt eine
langsame Vermehrung und Differenzierung der an ihrem neuen
Sitz ansdssig gewordenen Zellen. Man erkennt das Wachs-

82) Allerdings kénnen durch eine besondere Vorbereitung der Ge-
webe und der Kulturen auch Ventrikelstiicke von dilteren Embryonen
zur Pulsation gebracht werden. Die Ursachen, die einen Rhythmus
hervorrufen oder unterdriicken, werden in einer spiiteren Arbeit be-
sprochen werden.

Fig. 2. Wachstum der Zellen aus einem Stiick des Ventrikelmuskels
eines 12 Tage alten Hiihnerembryos. 8 Tage nach Herstellung des
Priparates. In Himatoxylin gefarbt.

tum an der Kernteilung und Protoplasmavermehrung der
Zellen bzw. des Synzytiums.

Rhythmische Kontraktionen. In dieser Periode
treten die rhythmischen Kontraktionen bei den
ausgewanderten und differenzierten Zellen auf.
Sie wurden in einem Fall schon am 5. Tag, in anderen Fallen
erst am 14. Tage des Kulturlebens konstatiert. Die rhythmisch
pulsierenden Zellen liegen stets im Fibringeriist und weisen.
ein durchsichtiges Protoplasma auf, welches vereinzelte Fett-
tropfen enthdlt. Unter 15 der nach der neuen Methode
angefertigten Praparate, bei denen das Gewebe fortwaéhrend
mit frischem Serum umspiilt wurde, fanden sich rhythmische
Kontraktionen neugebildeter Herzmuskelzellen bei 3 Prd-
paraten, wihrend nur bei 2 unter 150 der gewohnlichen
Hiangetropfenpriparate dies der Fall war. Das haufigere Vor-
kommen funktionierender Zellen in ‘den gr6ésseren Kulturen
hangt offenbar mit der bei diesen Kulturen langer andauernden
Periode des lebhaften Wachstums zusammen. Es wurden
naimlich Wachstumserscheinungen bei den grésseren Kulturen

a

bis auf den 30. Tag nach Herstellung des Pradparates kon-
statiert, wdhrend sie bei den gew6dhnlichen Héangetropfen-
kulturen zwischen dem 10. bis 18. Tage schon aufhdérten.
Wiederholte sukzessive Transplantation der Gewebekulturen _
verlangern zwar im ganzen die Dauer der funktionellen Tatig-
keit, aber obwohl solche Transplantation jedesmal wieder mit
einem neuen Auswuchs der Zellen begleitet ist, so ist doch
ein solches Verfahren keineswegs imstande, weder das Leben
der an Ort und Stelle neugebildeten individuellen Zellen, noch
die bei diesen auftretenden Perioden funktioneller Tatigkeit
zu verlangern.

Um auf die Bewegungserscheinungen, welche an isolierten
Zellen beobachtet wurden, etwas naher einzugehen, sei einiges
aus dem Protokoll der 12. Kultur angefiihrt. In diesem Prda-
parat hatte eine einzelne Zelle, welche weit vom urspriing-
lichen Gewebestiicke entfernt war, sich pulsierend bewegt,
und zwar mit einem Rhythmus, der von dem des Synzytiums
unabhangig war.

Die isolierte Zelle ist spindelférmig. Das eine Ende
ist abgerundet, wahrend das andere zwei ausgezogene Fort-
sdtze aufweist, welch letzteren grébere Fibrinfaden fest an-
haften. Die Lage des einzigen Kerns wird durch eine leichte
Ausbuchtung des Protoplasmas in der Nahe des runden Endes
verraten. Das feinkérnige oder maschige Protoplasma weist
einige perinukleadre, stark lichtbrechende Granula auf.

Die Phase der Kontraktion dauert betrachtlich langer als
die der Erschlaffung. Die Erschlaffung erfolgt plotzlich, und
erinnert an das Zuriickschnellen eines gespannten Gummi-
bandes. Bei der pl6tzlichen Entspannung der Zelle wirkt
moglicherweise die Elastizitaét der Fibrinfiden mit. Die Zelle
wird bei der. Kontraktion um ca. */s ihrer Lange verkiirzt,
wahrend ihr kurzer Durchmesser an allen Stellen scheinbar
vergrossert wird. Das Intervall zwischen den zwei Phasen
ist sehr klein. Bei mittlerer Vergrdsserung sind die Be-
wegungserscheinungen im Mikroskop leichter zu verfolgen und
man kann sie mittels Kinematographie aufnehmen, wie
Braus angegeben hat.

Am 14. Tage hat das Synzytium als Ganzes gleich-
massig pulsiert, wahrend am 15. und 16. Tage nur noch zwei
von einander getrennte Teile des Synzytiums pulsierten; zwar
war die Frequenz des Rhythmus gleich geblieben, doch trat
jetzt eine Phasenverschiebung ein, infolgedessen pulsierten die
zwei Teile nicht mehr synchron. Daher ist anzunehmen, dass
die zwischenliegenden Zellen nicht nur ihr spontanes Kon-

?

=

—- 10 —

traktionsvermoégen, sondern auch ihr Reizleitungsvermégen
eingebiisst hatten.

Zusammenfassung: Die MHerzmuskelzellen em-
bryonaler Hiihner kénnen, nachdem sie Teilung und Differen-
zierung ausserhalb des Organismus erfahren haben, ihre spe-
zifische Funktionstatigkeit sowohl als isolierte Zellen wie auch
als zusammenhingende Zellmassen wieder aufnehmen. Der
Rhythmus solcher Zellen stimmt mit dem des Herzens des
lebenden Tieres iiberein. Die rhythmische Bewegung wurde
nicht nur bei den ausgewanderten Herzmuskelzellen junger,
sondern auch bei denen der 14 tagigen Embryonen beobachtet.
Die Stiicke selber, die aus dem Ventrikel der alteren Em-
bryonen gewonnen sind, schlagen aber nicht, trotzdem die
aus solchen Stiicken isoliert ausgewanderten Zellen Kon-
traktionen ausfiihren.

Durch diese Unterstchung (st demnpaen
ein direkter Beweis fiir die myogene [Theorie
des Herzschlagves gebracht worden:

Reprinted from the American Journal of Physiology.
Vol. XXXI. — November 1, 1912. — No. II.

THE EFFECTS OF ALKALOIDS ON THE DEVELOPMENT
OF FISH (FUNDULUS) EGGS.

By J. F. McCLENDON.

[From the Embryological Laboratory of Cornell University Medical College, New York City,
and the U. S. Bureau of Fisheries, Woods Hole, Mass.]

HE goal of experimental embryology is the control of develop-

ment, notwithstanding the fact that the majority of attempts
in this direction have been failures. The embryo results from the
interaction between the egg and its environment, and we might ex-
pect that a specific change in the medium would produce a specific
change in the embryo. However, the organism is capable, to a great
degree, of maintaining constant conditions within itself. Take, for
example, the remarkable constancy in body temperature and com-
position of the blood of mammals.

One mechanism for the maintenance of constant chemical condi-
tions within the organism is evidenced in the remarkable semi-
permeability of living cells. Overton found that volatile anesthetics
and free alkaloid bases, which are rarely encountered by cells, penetrate
easily, whereas salts, with which cells are constantly in contact, do not
ordinarily penetrate. I observed that neithersalts nor anions penetrate
the Fundulus egg, but that kations outside may be exchanged for
those within.!

Herbst ? thought he had found a specific effect of lithium salts on
sea urchins’ eggs in the production of exogastrule, 7. e., gastrule in
which the archenteron is evaginated instead of invaginated. How-
ever, Driesch * produced the same results by a rise in temperature to
30° C. In this case the archenteron, or gut, sometimes shrank and
disappeared, producing a condition known as anenteria.

1 McCLeEnpon: this Journal, 1912, xxix, p. 295.

* Hersst: Zeitschrift fiir wissenschaftliche Zoologie, 1892, lv, p. 442, and
Mitteilungen der zoologische Station zu Neapel, 1895, xi, p. 136.

3 Driescu: Ibid., 1895, xi, p. 221.

131

132 J. F. McClendon.

x.

Gurwitsch * supposed that lithium salts produced a radially sym-
metrical gastrula in the frog’s egg. Morgan ® showed this not to be
the correct interpretation, but the chief characteristic of these embryos
is that the endodermal cells are not invaginated, and hence we might
call them exogastrule.

In opposition to the above statements, Bataillon® denies that
lithium or other salts or sugar act otherwise than osmotically, and
states that isotonic solutions all have the same effect on frog’s eggs.

Stockard ’ observed that lithium chloride causes an enlarged seg-
mentation cavity, and retards the down-growth of the blastoderm
over the yolk, in Fundulus embryos. He demonstrated that this is
independent of the osmotic pressure of the medium, and in a later
paper ® stated that these abnormalities are ‘‘specific for the lithium
ion in its action on this egg.”

On the other hand, I produced “lithium embryos” with sodium
chloride, calcium chloride, ether, acetone, and dextrose.®

Stockard produced cyclopic or one-eyed Fundulus embryos, and at
first thought theabnormality due to the specific action of the magnesium
ion,!” but laterobtained similar results by the use of volatile anesthetics,
and supposed them due to the specific action of anzesthetics.™

However, I obtained the same results, not only with several indiffer-
ent anesthetics, but with sodium chloride, lithium chloride, and
sodium hydrate, which are considered stimulating rather than anes-
thetic in their action.” I found the order of effectiveness of kations
(added to sea water) in producing cyclopia to be Mg<Li<Na. Since
Hedin * found the same order in the rate of diffusion of these ions
through dead ox gut, we may suppose their action in producing cyclopia
probably to be physico-chemical. This may be true also of indifferent
anesthetics, since I showed that their effectiveness in producing

4 Gurwirtscu: Archiv fiir Entwicklungsmechanik, 1896, iii, p. 219.
5 Morcan: Ibid., 1903, xvi, p. 691.

® BaTAILLON: Archiv fiir Entwicklungsmechanik, rgor, xi, p. 149.
7 STOCKARD: Journal of experimental zodlogy, 1906, iii, p. 399.

8 StocKARD: Ibid., 1907, iv, p. 165.

® McCLeEnpon: this Journal, 1912, xxix, p. 297.

0 StocKARD: Journal of experimental zodlogy, 1909, vi, p. 285.
SrocKARD: American journal of anatomy, 1910, x, p. 369.

® McCLenpon: this Journal, 1912, xxix, p. 289.

‘8 Hepin: Archiv fiir Physiologie, 1899, Ixxviii, p. 205.

Effects of Alkaloids on the Development of Fish Eggs. 133

cyclopia is proportional to their effectiveness in lowering the surface
tension of water, and also proportional to their toxicity. The con-
centrations of salts and anesthetics producing cyclopia are very near
the lethal doses, although lower than those producing “lithium
embryos.”

EXPERIMENTS.

The majority of drugs that have a specific action on the function
of parts of the human body are included in the old group of alkaloids.
Since the chemistry of many of these substances is unknown, the group
cannot be well defined, and we will use the name as originally applied
to substances of vegetable origin with basic properties. Many syn-
thetic substitutes of the alkaloids might well be included in the group.

I have tried a number of alkaloids on the eggs of Fundulus hetero-
clitus, and produced the same abnormalities with each of them. Some
experiments with glucosides were begun, but were cut short by the
unusually early close of the breeding season. Comparative studies
were made on the eggs of the sea urchin, Arbacia punctulata.

Of the alkaloids tried, caffeine and theobromine are xanthine bases,
the remainder being derivatives of pyridine and quinoline. Stovaine
(chlorhydrate of dimethy!aminobenzoylpentanol), a synthetic prod-
uct, was also used.

The alkaloids were usually made up in centi-molecular solutions in
sea water, and various strengths obtained by dilution. The free base
was used except in three cases. The hydrochloride of quinine was
chosen. The sulphates of strychnine and morphine were used, and
made up of half strength, since each molecule liberates two molecules
of the free base. These salts of the alkaloids have the advantage of
being more easily dissolved, and since sea water is alkaline, the free
base is completely liberated in the solutions. Overton showed that
alkaloids enter living cells only in the form of the free base, hence
they are effective only in alkaline or neutral solutions.

In the stronger solutions the whole embryo degenerates, but in
weaker solutions certain parts are affected more than others. Organs
which arise early may degenerate, those which appear later may fail
to develop. The circulatory system is the most affected, and may be
suppressed to a greater or less extent, or may develop and not function,
or may function for a while and then degenerate. The heart and

134 J. F. McClendon.

respiratory capillaries lie upon the surface of the yolk sac, and were
more carefully observed than were the vessels which are obscured by
the surrounding tissues of the embryo.

Ficure 1. FIGURE 3.

Figure 1,— Normal embryo of Fundulus heteroclitus, six days old. S, sub-intestinal
vein; D, duct of Cuvier.

Ficure 3.— Fundulus egg treated with ,'5 molecular caffeine, showing the degenerat-
ing embryo one week old.

Ficure 4.— Front view of embryo from ;% caffeine, one week old. The heart beat,

but the blood did not circulate.

A glance at the normal embryo will be useful for comparison. Fig. 1
shows the yolk sac circulation in a six-day embryo, the capillaries on
the ventral side being indicated by dashed lines. The caudal vein
passes on to the yolk sac as the sub-intestinal vein (S), and breaks

FicureE 2.— The same embryo as Fig. 1, just hatched.

up into capillaries. The ducts of Cuvier pass out of the embryo
and break up into capillaries, which anastomose with those from the
sub-intestinal vein. This capillary plexus is reunited at the venous
end of the heart, in front of the head of the embryo. It should be
noted that in early stages the ducts of Cuvier draw their blood from
the dorsal aorta through temporary connections.

As the embryo develops, the yolk is absorbed, and the capillaries on
the yolk sac are gradually transformed into three large veins, the
continuations of the ducts of Cuvier and the sub-intestinal vein. A
transition stage is seen in an embryo just hatched (Fig. 2). The

Effects of Alkaloids on the Development of Fish Eggs. 135

right duct of Cuvier (D) is shown completed, but the sub-intestinal
vein (S) is connected with the heart by four parallel veinlets, the
remains of the capillary plexus. The anastomoses between the sub-
intestinal vein and ducts of Cuvier have disappeared.

In the stronger solutions of alkaloids the embryos begin to develop
normally, but sooner or later the cells begin to be loosened’one from

Ficure 5. FIGURE 6. FIGURE 7.

Ficure 5. — Same embryo as Fig. 4, ten days old.
FicurE 6.— Embryo from %& caffeine solution, eleven days old. -
Ficure 7.— Cyclopic monster from 7¥, nicotine solution, four days old.

another, a condition called by Roux “‘framboisea.’’ Such an embryo
may live a long time, but gradually undergoes de-differentiation.

In Fig. 3, which represents such an egg a week old, the stippled area
represents the embryo. The spots with blunt processes represent
black chromatophores, and those with slender processes, red chroma-
tophores. The blister on the yolk is the swollen pericardial cavity,
and is the only means by which the head end of the embryo may be
located.

Fig. 4 represents an embryo of the same age from a weaker solu-
tion, viewed from the front. The eyes are represented by the small
circles. The semicircular areas, lateral to the eyes, are the distended
ear vesicles, whereas the almost circular area reaching from the eyes
to the dashed line across the middle of the yolk, represents the dis-
tended pericardial cavity. The elongate body, extending from the
head ventralward, is the heart. It is beating, but no blood circulates,
since no hollow vessels are connected with it. Erythrocytes, repre-
sented by stipple, lie in the arterial end of the heart. The lower or
venous end of the heart is covered with red chromatophores.

In this same embryo three days later the eyes are degenerating, the
left eye being represented by merely a thin smear of retinal pigment

130 J. F, McClendon.

(Fig. 5). The number of chromatophores on the ventral side of the
pericardium and on the heart is greater than in a normal embryo,
Owing to the swelling of the pericardial cavity, the heart is greatly
elongated and its middle portion is transformed into a solid cord.
Sometimes but one eye completely degenerates, resulting in a con-
dition which I have called secondary monophthalmia asymmetrica;

Ficure 8. FIGURE 9,

FicurE 8.— The same embryo as Fig. 7, sixteen days old. ,
Ficure 9.— Monstrum monophthalmicum asymmetricum, sixteen days old, from My
nicotine solution.

or the two eyes partially degenerate and then fuse together, secondary
cyclopia.

Often the anterior region of the head disintegrates, and the cells
wander away and become scattered over the pericardium. Such an
embryo is shown in Fig. 6, and is atypical only in the fate of the eyes.
The heart formed, but did not beat. The stippled area in the tail
region represents a mass of erythrocytes. The pericardium was
greatly distended and pressed against the ventral side of the head, to
which it adhered. The anterior portion of the head disintegrated,
and the eyes contracted into two spherical masses blackened by
retinal pigment. By the eleventh day the eyes had fallen through
the pericardial cavity and become grafted on to the heart.

Only one case of primary cyclopia occurred. This embryo, when
four days old (Fig. 7), was apparently normal except for the single
dumbbell-shaped eye on the ventral side of the head. The same
embryo, sixteen days old, is shown from the front in Fig. 8. Although
it had been removed from the nicotine solution when thirty-six hours
old and had remained in pure sea water, frequently changed, for two
weeks, defects began to appear in the circulatory system, and the
circulation ceased entirely long before death. As shown in Fig. 8,

Effects of Alkaloids on the Development of Fish Eggs. 137

the pericardial cavity is distended and its contents turbid, and in-
clude a mass of erythrocytes around the venous end of the heart, rep-
resented by stipple.

The only case of primary monophthalmia obtained is shown at the
age of sixteen days in Fig. 9. Except for the lack of the right eye,
it is apparently normal. Even the right eye socket has developed
in an apparently normal manner, but is covered by skin containing
chromatophores.

TABLE I
Caffeine . . 50 80-200 Quinine .. 200 400
Atropine . . 90 ~=—- 100 Strychnine . ae 100-1600
(Saturated)
Brucine. . . se AQ OK SAEMERECH Bie Bere ety. oc Fa. mous wt
Nicotine . . 350 400-700 Stovaine . . 800 1600

The alkaloids, even in very weak solutions, retard development.
The toxic limits, and concentrations of solutions causing the abnor-
malities described above, are given in Table I. A saturated solution
of theobromine produced no monsters, due to its very slight solubility,
but, from comparative studies on the egg of the sea urchin, we may
class it with caffeine.

To give a uniform basis for comparison, the data of only those ex-
periments in which the embryos remained in the solutions thirty-six
hours, beginning with the two-cell stage, are used. The “toxic
limit” is the solution in which nearly all of the eggs are dead at the
end of thirty-six hours. Each number in the table is the denominator
of a fraction (of a molecular solution) whose numerator is 1. The
middle column gives the toxic limits, and the last column gives the
concentrations which suppress the circulation in various degrees, and
cause the other abnormalities described above.

Comparative experiments were made on the eggs of the sea urchin,
Arbacia punctulata. These eggs are more easily obtained, develop
more rapidly, and are more sensitive to changes in the medium than
are Fundulus eggs. All of the alkaloids, as well as stovaine and
digitalin, produced the same abnormalities.

The strengths of the solutions are shown in Table II. The eggs
were not removed from the solutions and returned to pure sea water,
as in the case of the Fundulus eggs. The solutions of digitalin are

138 J. F. McClendon.

percentages, in case of the others the number represents the denomi-
nator of a fraction (of a molecular solution) whose numerator is 1.
In stronger solutions than those listed in the table, embryos died
in segmentation stages. Even in those in the table development was
enormously retarded, and the yolk granules dissolved more slowly,
although ciliary activity did not appear to be reduced. Whereas

TABLE II.
Caffeine .. 500 800 Nicotine . . 3,200 6,400
Theobromine ......... Saturated Quinine. . 50,000 55,000
Atropine... 3,200 25,600 Strychnine. . 25,600 51,200
Brucine. .-. 12,800 51,200: Wa ISE SRE) ee
Cocaine :' . : 6,400 25,600 Stovaine . . 12,800 102,400.
Morphine . . 800 6,400 Digitalin . . .0004% -.0001%

normal embryos, if not fed, starve to death in a few days, some
embryos in the alkaloid solutions live several weeks. After the first
few days a distention of the body cavity commences and may con-
tinue until the ectoderm becomes a very thin-walled vesicle.

In the solutions listed in the middle column plutei were not pro-
duced, although rudiments of the skeleton, in the form of tri-radiate
stars, sometimes appeared. Some exogastrula were produced. In
case normal invagination of the endoderm did take place, the gut was
never normally differentiated, but usually degenerated into a solid
mass of cells.

In the solutions listed in the last column plutei were formed, but
there was an early disarrangement of the mesoderm cells, so that the
resulting skeleton was abnormal. Since there was an enormous num-
ber of forms of the skeleton, they cannot be described here.

CONCLUSIONS.

It has been shown above that the very different organic compounds
used, belonging to boththealiphatic and the carbocyclic series, although
included in the old class of alkaloids, have the same morphological
effects on the eggs of Fundulus heteroclitus. Loeb has obtained the
same abnormalities in solutions of potassium cyanide and has reared
similar monsters from eggs fertilized with foreign sperm.

Effects of Alkaloids on the Development of Fish Eggs. 139

It might be supposed that these effects follow any injury to the
Fundulus egg. The distention of the pericardium follows the appli-
cation of various salts. I have produced it in frog’s embryos by
mechanical injury. The distention of other serous cavities sometimes
follows the application of certain salts and anesthetics. Loeb pre-
vented the heart beat with potassium salts. Some embryos in am-
monium salts were observed by Stockard never to develop a heart
beat, andinsome inmagnesiumsalts thecirculatorysystem degenerated.

It may be possible that the same abnormalities can be produced
by any chemical treatment. However, the quantitative data show
striking differences in the effects of different substances. Using solu-
tions of equal toxicity, almost roo per cent of embryos in ethyl alcohol
may show primary defects in the eyes, whereas such defects are seen
in not more than one tenth of x per cent of embryos treated with alka-
loids. On the other hand, the abnormalities described in the circula-
tion may occur in nearly 1oo per cent of embryos treated with
alkaloids.

Thousands of eggs of Fundulus heteroclitus were placed in solu-
tions of each of the alkaloids enumerated, and the eyes of each embryo
were carefully examined, but only one case of primary cyclopia and
one of primary monophthalmia asymmetrica were observed. These
two occurred in the same batch of eggs treated with nicotine. I sub-
sequently used this same concentration of nicotine on the eggs of
many different females without reproducing these abnormalities.

Stockard obtained very different percentages of cyclcpia in eggs
treated with magnesium salts during different seasons or parts of
seasons. He once thought this due to accidental variation in the
concentration of the solutions. This could not have been the case
at least in my experiments, as I used a finely graduated series of
concentrations extending a great distance on each side of the apparent
optimum for producing cyclopia. In numerous experiments covering
an entire season, I failed to obtain as high a per cent of cyclopia with
magnesium chloride as recorded by Stockard. By careful measure-
ments of specific gravity, I found that the varying results were not
due to differences in density of the sea water. The water I had been
using was obtained directly from the sea in glass vessels, but as a
control I used sea water drawn from the same pipe as that used by
Stockard. This was repeated many times, and it was demonstrated
that heavy metals in the water did not account for the differences.

140 J. F. McClendon.

The cause of the varying results must be that different batches of
eggs have not the same tendency toward cyclopia, just as the individ-
ual eggs laid by the same female at the same time vary in this respect.
Perhaps some Fundulus eggs would produce cyclopic embryos without
any laboratory treatment, as I have found many cyclopic smelt _
embryos in the natural breeding places. It would not be safe, there-
fore, to draw conclusions from two individuals in many thousands.
Attention should be directed rather to the quantitative data.

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Reprinted from THE JourNAL or BroLocicat CHEemistry, Vol. XI, No. 4, 1912

ECHINOCHROME, A RED SUBSTANCE IN SEA URCHINS.

By J. F. McCLENDON.

(From the Embryological Laboratory of Cornell University Medical College,
New York City, and the U. S. Bureau of Fisheries, Woods Hole, Mass.)

(Received for publication, March 30, 1912.)

INTRODUCTION.

My interest in echinochrome arose from studies in permeability.
In the same way that haemolytic agents cause haemoglobin to
leave the red blood corpuscles, so do cytolytic agents cause echino-
chrome to leave the cells containing it. R. Lillie is of the opinion
that this is due to the action of the cytolytic agent in increasing the
permeability of the cell surface.

In the elaeocytes, wandering cells of the body fluid of Arbacia
punctulata, the cytoplasm is crowded with spherical chromatophores.
Some of these may be colorless, but usually they are colored bright
red with echinochrome. Similar chromatophores, though not so
close together, occur in the eggs. In the unfertilized egg they are
evenly distributed throughout the cytoplasm. But after fertili-
zation, the chromatophores all migrate to the surface within half
an hour. During cleavage of the egg, they are massed in the cleav-
age furrows. The pigment occurs also in the test of this sea urchin,
and gives the animal the characteristic color, which varies from a
bright red (especially in young individuals) to a dark red, and may
be almost black in old specimens.

In reference te the fact that the pigment may be caused to leave
the chromatophores and pass into the cytoplasm and thence into
the medium, the following questions may be asked: (1) How is
the pigment held in the chromatophores? (2) What is its fune-
tion? (3) What is its chemical nature? The present paper is
concerned with these questions.

435

436 Echinochrome

HISTORICAL.

Echinochrome was studied spectroscopically by McMunn,! who found it in
the elaeocytes of the sea urchins, Strongylocentrotus lividus, Amphidotus
cordatus, Echinus esculentus? and E. sphaera. The spectrum showed faint
absorption bands, which varied with different solvents and different reac- -
tions of the same solvent. McMunn thought that he noticed changes in the
spectrum on the addition of powerful reducing agents, such as stannous
chloride, and concluded that echinochrome functioned as an oxygen carrier.
However, the absorption bands in its spectrum are difficult to make out
except in absolute alcohol (or glycerine) and in this solvent I observed that
stannous chloride caused a precipitation of the pigment, which interfered
with the examination.

A. B. Griffiths? attempted an elementary analysis of the substance. He
dried the elaeocytes and extracted them with chloroform, benzol or carbon
bisulphide. On evaporation of the solvent he analyzed the substance with-
out further purification, although evidently it contained many impurities.
From four analyses, he deduced the formula Cio2Ho9Ni2FeS:012, which
would make C = 67.8 per cent, H = 5.5 per cent, and N = 9.3 per cent.
He states that on boiling with mineral acids it is transformed into haemato-
porphyrin, haemochromogen and sulphuric acid (KE + acid = 2C3,H34N,0;
+ C3,H3;NFeO; + H2SO,). Griffiths agrees with McMunn that echino-
chrome is an oxygen carrier, and states that the oygen is held rather firmly,
and in nature is removed only by the reducing action of the cell containing
the pigment. _

EXPERIMENTAL.

The pigment in the elaeocytes, eggs and tests of Arbacia, shows
no absorption bands, but after extraction it shows very similar
bands in its spectrum to those described for echinochrome by
MecMunn. He published drawings of the spectra and measured the
wave lengths corresponding to the edges of the bands. It is well
known that bands become broader as the solution is more concen-
trated, and for that reason I measured the wave length of aline of
the spectrum corresponding as nearly as could be determined to
the center of each band. By taking the mean between the wave
lengths of the edges of the band in McMunn’s data I have com-
pared his with mine. The discrepancies may be accounted for,

1McMunn: Quart. Journ. Micro. Sci. (2), xxv, p. 469, 1885; xxx, p. 51,
1889.

2 Griffiths: Compt. rend. soc. biol., exv, p. 419, 1892; Proc. Roy. Soc. Edin-
burg, xix, p. 117, 1892; Physiology of the Invertebrata, New York, 1892; Respira-
tory Proteids, London, 1897.

J. F. McClendon 437

first by the fact that the mean is not the exact center of the band in
a prism spectrum, and secondly there is a personal equation in
observation. I found the pigment extracted from elaeocytes, eggs
or tests to give about the same spectra, though a few isolated obser- -
vations seemed to vary. These might have been due todecom-
position products with different spectra.

ABSOLUTE ALCOHOL H:0
ETHER

Neutral +Hcl] | +NHs +HCl | -+-NHs

My data... 5296 4844 | 5504 | 5302 4844 5296 4844 | 5154 4844 | 5296 4844 | 5154 4844

MeMunn .. 5512 | 5128 | 4848 5370 | 4998 | 5205 | 4848 |
| { Hi

Neither McMunn nor Griffiths succeeded in crystallizing echino-
chrome. Dr. A. P. Mathews had observed that on the addition of
iodine in potassium iodide (KI) crystals form easily. In 1910
I obtained quantities of these crystals, but did not succeed in recrys-
tallizing them without great loss by the formation of amorphous
masses. The iodine compound in absolute alcohol showed an
additional, but very dim band in the spectrum (wave length 5628
or 5696). It crystallized in red or orange needle-like crystals,
triangular in cross section, sometimes rhombic in side view and
often forming rpsettes. They were but slightly soluble in water
unless hot or containing acid, soluble in absolute alcohol (the
rhombic crystals seeming more soluble than the needles) and slightly
soluble in ether. If a solution in water is shaken with ether the
latter is not colored. If an alkali is added to the KI; solution no
erystals are formed (due to combination ofthe base with the
echinochrome) but HCl does not prevent their formation.

Some of this iodine compound which was kept for several months
in a dry state became more soluble in ether and crystallized in flat
thin, red or orange rhombic plates. Perhaps the substance had
decomposed with the liberation of iodine, for I succeeded in crys-
tallizing the mother substance and obtained the same plates, in
addition to red or orange needles, never triangular in cross section,
but sometimes forming rosettes.

I extracted echinochrome from the tests with strong, slightly
acidulated alcohol and purified it by repeated precipitation with

438 Echinochrome

alkali and solution in acid alcohol, and filtration. Finally I
dissolved the precipitate in water plus HCl, filtered and shook the
solution with ether. The ether did not remove all of the echino-
chrome and thé formation of haptogen membranes caused much
loss of material. The ether was evaporated at room temperature,
as heat seemed to decompose the substance. Occasionally a few
crystals formed at the edges of the solution but the main mass of
the residue was amorphous.

The next season (1911) I tried to purify echinochrome without
the use of acids or alkalies. The body fluid of the sea urchins was
allowed to clot and the elaeocytes thus obtained were placed
directly into acetone, which extracted the pigment. The extract
was filtered and evaporated at room temperature. The residue
was washed with carbon tetrachloride (which does not easily dis-
solve echinochrome) to remove fats, and again dissolved in the
smallest quantity of acetone and filtered to free it from traces of
lecithin. This solution was evaporated, dissolved in absolute
ether and filtered to remove salts, evaporated to constant weight
and analyzed by Dennstedt’s method. A mean of two analyses
gave: C = 51 per cent, H = 7.7 per cent. The echinochrome
purified by precipitating with alkali gave C = 53.3 per cent,
H = 4.4 per cent, N = 1.5 per cent. The nitrogen was deter-
mined by Kjeldahl’s method and therefore may not be reliable,
since the constitution of the molecule is unknown. Traces of
sulphur and phosphorus, possibly due to impurities were found, but
no iron. The ether-soluble crystals from spontaneous decomposi-
tion of the iodine compound gave C = 57.9 per cent, H = 6.5
per cent. :

It was stated above that echinochromeé is precipitated by alkalies
in alcohol. I precipitated echinochrome with NaOH in 965 per
cent alcohol and washed in the same alcohol to remove the excess
of NaOH. From the amount of NaOH that was neutralized by
the pigment I concluded that it combined with from 18 to 25 per
cent of Na. Analysis gave C = 31.5 per cent, H = 6 per cent,
Na = 19.5 percent. Therefore we may say that the echinochrome
behaves as an acid, or else is amphoteric. The former view is

* Alkali does not precipitate it in water; the particular base was immate-

rial, ammonia was added but the presence of sea salts allowed the libera-
tion of other bases.

J. F. McClendon 439

supported by the fact that on passing an electric current through
the aqueous (colloidal) solution, the echinochrome shows a nega-
tive charge (is anodic) and again, if histological sections are placed
in such a solution the acidophile portions are stained more strongly °
than the remainder. In fact its behavior is very similar to that of
a weak solution of eosin, except that it is very easily washed out
by alcohol.

However the substance is probably amphoteric (the acid charac-
ter being stronger than the basic) since its aqueous solution is
precipitated by phosphomolybdic and phosphotungstic acids but
not by tannin.

From the analyses given above it would seem that no one has
succeeded in obtaining echinochrome in a reasonably pure state.
It is very unstable and probably breaks up into a host of decompo-
sition products all having practically the same spectrum. [If it is
kept in the dry state for a great length of time, or is evaporated
on a bath not over 50° for a shorter time, part of it becomes insol-
uble in ether but not in alcohol.

When heated in the combustion tube it first melts, then boils
and sublimes as crystals on the top of the tube, then very soon
turns. brown and chars. After being crystallized from a solution
in ether the crystals often become smaller and irregularin outline.
Perhaps the crystals evaporate or lose water of crystallization, but
I think that both these possibilities are improbable. The crys-
tals may decompose into an amorphous substance.

On first obtaining crystals, I feared that they were crystals
of some other substance merely colored by echinochrome, but this _
seems impossible from later observations.

In extractions made for the purpose of studying the lipoids in-
Arbacia eggs, red or brown substances (echinochrome or its decom-
position products) appear in every fraction, rendering analysis
difficult and indicating the instability and wide solubility of the
substance.

In order to test the statement that it is an oxygen carrier I
separated the cells from 50 ec. of body fluid- by the centrifuge,
and mixed them with sea water to make 50 ce. This suspension,
and 50 cc. of sea water as a control, were placed in two similar
graduated tubes. The air was pumped out for six hours (until the
water boiled), air was then admitted and the tubes sealed. They

440 Echinochrome

were shaked one-half hour and the volume of air measured at
atmospheric pressure. ‘The suspension had lost 1.25 ce., the con-
trol only 0.8 cc. In another experiment the suspension lost 0.95
ce. and the sea water 0.8 ec. It was thought that in the absence of
oxygen the cells would take the oxygen from the echinochrome.
However no color change could be observed with the naked eye
or the spectroscope, and the greater absorption of air by the sus-
pension may have been entirely due to oxidation in the cells. In
similar experiments, with an aqueous solution of the pigment, and
distilled water for a control, and using pure oxygen, the two tubes
eave the same absorption, as shown by two examples:

Oxygen absorbed by HO) o.2hd) ob) ieee ee ca f
Oxygen absorbed by echinochrome.............-.)++.-.s.5a6

The question, how echinochrome is held in the chromatophores,
cannot be fully answered. The chromatophores when free from
pigment are highly refractive and stain strongly with the intra-
vitam stain, neutral red, and when fixed they stain strongly with |
Delafield’s haematoxylin, indicating a lipoid nature. The pig-
ment may be in solution in the lipoid.

The fact that the spectrum is different (shows no bands) in
life from the spectrum of the extract may indicate chemical com-
bination of the pigment with the chromatophores. The fact that
echinochrome stains acidophile tissue may show a possible mode of
_ such combination, if it be found that the chromatophores contain
bases. However I do not think we can rely on the spectroscopic
evidence, for the absorption bands are very faint in aqueous solu-
tion unless it be alkaline, and the cells containing the pigment
interfere with the passage of light and make the observation diffi.
cult. I have never seen absorption bands in echinochrome ex-
tracted from the fresh cells with distilled water. The same state-
ment is made by McMunn. If the substance is held by chemical
combination why does it come out so easily?

The same argument may be made against the possibility that
the echinochrome is held in the chromatophores because it is more.
soluble in them than in water. When the cell is stimulated me-
chanically or chemically the pigment comes out of the chromato-

J. F. McClendon AAI

phores with explosive rapidity. The cell need not be killed to
accomplish this. The mere act of normal fertilization causes
some of the chromatophores in the egg to lose their pigment.

The only alternative hypothesis I know of is, that the pigment is.
manufactured in the chromatophore, and cannot normally get out
because the surface of this body is impermeable to it. An increase
in permeability of the chromatophore allows the pigment to escape.
Such an increase in permeability might be due to an aggregation
change in the colloids of the limiting membrane or surface film.

Eehinochrome is held in the chromatophores of the sea urchin’s
cells probably in the same way that chlorophyll is held in the -chro-
matophores of the green plant cell.

x
x
-
’
“
’
.
ee

Reprinted from Tur ANATOMICAL REcorRD, Vol. 7, No. 2,
February 1913

PREPARATION OF MATERIAL FOR HISTOLOGY AND
EMBRYOLOGY, WITH AN APPENDIX ON THE AR-
TERIES AND VEINS IN A THIRTY MILLIMETER PIG
EMBRYO

J. F. McCLENDON

From the Anatomical Department of Cornell University Medical College, New York

THREE FIGURES

The essential of a good course in histology or embryology is
good material. Fresh human material should never be allowed
to go to waste, but it may be at times very inconvenient to put it
up in a variety of fancy fixing fluids.

Perhaps the best general cytoplasmic fixer is formalin of 10 to
20 per cent (4 to 8 per cent formaldehyde). If material so fixed
is not soaked too long in alcohol of high concentration, it may be
used as fresh tissue in special technique to show fats or mitochon-
dria. In fact the formaldehyde alone makes unsaturated fats and
lipoids less soluble in clearing fluids. On the other hand, if the
washing in water is omitted, the structure of resting nuclei is -
well enough preserved for ordinary purposes.

Commercial formalin contains formic acid, which, although
developing a more beautiful nuclear structure, may begin to
cytolyse the more delicate cells before they are sufficiently fixed
by the formaldehyde. This is especially noticeable in erythro-
cytes—haemolysis, or escape of haemoglobin, occurring in parts
of the tissue. Furthermore, acids swell fresh white fibrous tissue.
It seems worth while, therefore, to neutralize the formol, and this
may easily be done by adding slack lime (CaCO;) or magnesia,
and filtering.

Doctor Ferguson first called my attention to the fact that kid-
ney swells in many fixing fluids, whereas it is commonly supposed

51

THE ANATOMICAL RECORD, VOL. 7, NO. 2

52 J. F. McCLENDON

that the majority of tissues shrink a little. Death: of isolated
cells as seen under the microscope may be accompanied by swell-
ing (cytolysis) or contraction. In every case, an increase in per-
meability to some substances occurs, but I found that during the
early stages of cytolysis of the sea urchin’s egg, it remains very
impermeable to salts. Dead animal or plant membranes are more
permeable to water than to dissolved substances, but apparently
some living cells are impermeable to water. The Fundulus egg,
if transferred from sea water to distilled water, does not burst,
though it is certainly not capable of resisting the enormous
osmotic pressure of its internal salts. Since I found this egg to be
impermeable to salts, it must also be impermeable to water (it is
permeable to kations, but for every kation that comes out, the
electrical equivalent must goin). If such a cell became, on death,
permeable to water, the osmotic pressure of its internal dissolved
substances might cause it to swell. If a Paramoecium be killed
by an ordinary fixing fluid, even though it be hypertonic, the
protoplasm first coagulates, then the whole animal swells a little.
This may be what happens to some tissue cells, and I found that
it is not always prevented by the addition of 0.9 per cent sodium
chloride to the fixing fluid. Therefore I supposed the swelling due
to the osmotic pressure of some contained substance of large
molecule, and experimented with the addition of cane sugar to
neutral formol. By this means the cytolysis of adult convoluted
nephric tuhule cells is prevented, and the general fixation is good
except that some nuclei may be slightly shrunken. This fluid
may be used for all adult tissues and embryos, and is easily pre-
pared as follows:

Formol). .. 0525. sce sae ae ee on be ee 100-200 ce
Cane sugars {...5..0/0) .. Se ee ee 20-40 grams
Slack lime (CaC@;) or magnesia: ......5)..::.2..) 0 ee about 1 gram

Water to make 1 liter.

If the shrinkage of a few nuclei is very objectionable use only
20 grams of sugar. This fluid has the advantage that tissues and
embryos float in it and therefore do not become distorted.

If the whole kidney of a fetus be fixed in the above mixture or
any other fixing fluid, the cells of the convoluted tubules will

MATERIAL FOR HISTOLOGY AND EMBRYOLOGY 53

swell until they fill the lumen. This brings us to a well known
point that is often neglected. Tissues should be cut into as thin
slices or pieces as is practicable and the cells not injured in the
cutting. Fetal tissues are especially delicate. They should be
cut with a very sharp thin blade and lifted on the blade into the
’ fixing fluid.

Many workers object to formalin because it ‘‘ causes”? a homo-
geneous appearance to protoplasm. ‘The ultra microscope has
shown that, aside from evident granules, living protoplasm is
homogeneous, contrary to Biitschli and others. There are per-
sons who now accept formalin for cytoplasmic fixation but say
that it ‘‘does not fix nuclei well.’”’ Some structures may be seen
in living nuclei. I have studied many nuclei with high powers
and with the ultra microscope, yet I cannot decide what form of
fixation corresponds most closely to the living structure. Both
cytoplasm and nucleus of a living erythrocyte of a frog is homo-
geneous when examined in serum or uncoagulated plasma with
the ultra microscope. Sooner or later bright points or clouds
appear on or in the nucleus, but this is usually associated with
change of nuclear form and is evidently due to injury.

Formaldehyde not only does not coagulate protoplasm but
renders it more difficult to coagulate. It also makes lipoids less
soluble in clearing fluids. However, I find an after-treatment
with Miiller’s fluid or some other oxidising fluid necessary for
the preservation of lipoids, the amount of oxidation necessary
depending on whether mitochondria, myelin or fats are studied.

Ordinary staining depends on the fact that all protoplasm
treated with acid, stains with acid dyes, whereas certain parts
take also basic dyes. Many staining solutions contain free acid,
but tissues stain more quickly if they are previously treated
with acid. For this reason we put everything into the formol
mixture and after a few hours transfer part of it to Bouin’s
fluid. This tissue is finally stained on the slide in haematin and
eosin. The alum haematin lake is usually so strong that it
stains in three minutes, but the eosin is so much diluted that
twelve hours are required to stain and in this time smooth mus-
cle stains less intensely than white fibrous tissue The acid in

54 J. F. McCLENDON

Bouin’s fluid causes the tissue to stain more brilliantly but if
the fresh tissue is put into Bouin’s fluid the blood in some of the
vessels will be laked. Part of the material is transferred from
the formol mixture to Miiller’s fluid and subsequently stained
with iron hematoxylin to show the lipoids (mitochondria, etc.).

Ordinarily, the student is shown two dimensions of a piece ©
of tissue or embryo, and left to imagine the third. Though
whole mounts of chick embryos are handed out, cleared pig em-
bryos, and blocks or thick sections of certain tissues are even
more useful. For a solid mount, the object should be placed
in a dish of balsam or damar dissolved in benzol and protected
from dust until it evaporates down to sirupy consistency, then
mounted in the usual way. By this means the necessity of
rings or other supports to the cover glass is avoided, and drying
out or great shrinkage prevented.

All of the solid mounts turn yellow with age, but a number
of highly refractive fluids may be obtained that are colorless.
These are listed, with their refractive indices, in Landolt-Born-
stein; Behren’s Tabellen; and Lee’s Vade Mecum. The higher
the refractive index the better, for if in any case a lower index
is desired, this may be obtained by the addition of paraffin oil
or xylol (or water in case of aqueous media). It may be noted
here that, whereas the process of clearing in a mixture of oil
of wintergreen (Gaulteria) and benzyl benzoate has been patented
in Germany and is widely known under the name of the patentee,
wintergreen was first used by Stieda in 1866, and the synthetic
oil (methyl salicylate) recommended by Guéguen in 1898, and
is noted in various books on technique.

Methyl salicylate is permanently colorless, and comparatively
inexpensive, and ideal for a fluid mount. If rings are cemented
on slides with shellac or liquid glue and allowed to dry, they
are not loosened by the oil. Paper rings soaked in shellac or
glue will do, but rings may be cut from lead pipe with an ordi-
nary saw or a bone saw if the proper size of glass rings are not ~
at hand. The shellac must be dry before adding the oil, which
must be free from alcohol. I prefer glue.

If the tissue is hardened in alcohol, thick sections may be cut
free-hand. Thick sections are often better unstained, especially

MATERIAL FOR HISTOLOGY AND EMBRYOLOGY 55

if injected, and much detail may be made out by partly closing
the diaphragm of the microscope. If stained with very dilute
haematin containing much acid, connective tissue is colorless and
cytoplasm nearly so, whereas nuclei may be readily distinguished.
In this way blood vessels and glands in areolar tissue are caused
to stand out sharply.

Whole mounts and thick slices are especially useful in embry-
- ology, and are a necessity unless one is contented with teaching
the third dimension with models. The larger the embryo, the
more attention must be paid to the clearing medium in order to
distinguish internal structures. Methyl salicylate is admirable
for pig embryos of all sizes and even for small fetuses. I found
ethyl salicylate to be as good if not better, but it is more expen-
sive. Canada balsam has about the same refractive index
("D = 1.535) as methyl salicylate (“D = 1.536), but darkens
with age. :

Embryos may be placed directly from absolute alcohol, benzol,
xylol, toluol or chloroform into methyl salicylate, but in order
to obtain the proper refractive index, the preliminary fluid must
all be removed. This may be evaporated, or washed out with
more wintergreen. Benzol is to be recommended because it is
cheapest and evaporates out most easily. The evaporation may
be hastened by an air pump, which also removes any air bubbles
that may get into the specimen. These bubbles expand and
are absorbed after the pump is disconnected, or by successive
pumpings. An ordinary air pump will cause the benzol and air
to boil out. A water-suction air pump (aspirator) will suffice
but a float valve and safety bottle should be interposed between,
the pump and the specimen to prevent the back flow of water.
An exhaustible desiccator is convenient for holding large em-
bryos while they are being pumped out. If the cover is well
ground, the oil will seal it sufficiently, and vaseline should not
be used.°

Most of the internal organs may be distinguished in unstained
embryos by cutting down the light. The individual cells of
mesenchyme, cartilage and blood may be seen; the cellular struc-
ture of the neural tube is indicated by radial striations and the

56 J. F. McCLENDON

larger nerves appear as bundles of fibers. Some organs in smaller
embryos are made more distinct by staining with very dilute
alum haematin containing a large amount of acid.

Even in quite small embryos, many of the blood vessels may
be traced by the blood cells, and the large empty veins followed
as cavities. ‘ However, with the smaller vessels this becomes more
laborious than serial sections. On the other hand, the injection
of small embryos for class use means quite an outlay of time..
Therefore, it seemed necessary to find some way to fix the haemo-
globin, and keep the vessels full, in order to distinguish the
vessels by the color of the blood. I found that the same method
that prevents the cytolysis of nephric tubule cells prevents haemolysis.

Living embryos are obtained, the amnion opened, the placenta
squeezed to force the blood into the embryo, and the umbilicus
tied or clamped. Artery clamps are too strong and pinch off
the cord. (I made clamps out of wire (lower part of fig. 1) in
order to avoid tying so many cords at the slaughter house.
The clamp may be removed in half an hour and used again.)
The embryo is dropped into the neutral-formol-sugar mixture
described above, and left until thoroughly fixed. In case of a
fetus, part of the skin should be-torn off after the superficial
blood vessels are fixed, to insure penetration of the formaldehyde.
A hole may be made in the skull by slicing off a small piece
tangentially or by a sagittal cut near (to the right of) the median
plane. Large fetuses, unless skinned completely, will have to
be scraped to remove the pigment layer.

Transfer the specimen after washing, or directly, from the
fixing fluid to aleohol of about 70 per cent. After they have
hardened in 80 or 95 per cent alcohol it is well to split the large
specimens by a sagittal cut a little to the right of the median
plane with a very thin bladed knife. The dehydration with
higher alcohols should be slow enough to prevent shriveling.

By this method the blood retains its color, and although it
does not take the place of injection, it is a great help to the
student. I have inserted three figures to show what can be seen
in such specimens.

MATERIAL FOR HISTOLOGY AND EMBRYOLOGY D

ma
oH & )S

<>

Fig. 1 Left half of a pig embryo 7 mm. long after clearing. The veins are
black, the arteries cross-striated, the 5th, 7th, Sth, 10t h and 11th cranial nerves
are longitudinally striated, the notochord is represented by a heavy line and the
fore gut by a dashed line. The sinuous line ventral to the embryo represents a
wire (partly open) clamp used in clamping the umbilicus.

58 J. F. McCLENDON

Figure | represents a pig embryo about 8 mm. long. The
nerve tube, fore gut, mesonephros, liver, heart, eye and ear are
clearly seen. ‘The arterial system and part of the cardinals and
subeardinals can be distinguished. The notochord is distinct,
and the 5th, 7th, 8th, 10th, and 11th cranial nerve roots can be
made out. Figures 2 and 3 are described in the appendix.

I have prepared hundreds of pig embryos and fetuses in this
way, and also injected many with india ink and cleared them
in wintergreen oil. A completely injected fetus can only be
studied in comparatively thin (freehand) sections. Various de-
grees of partial injection are very useful to show the larger ves-
sels, but these may be seen in the uninjected fetuses. The left
side of an uninjected fetus which has been cleaved a little to the
right of the median plane, will show the general circulation,
except in the liver. The larger vessels in the liver may be seen
by removing the lateral portions and passing a strong light
through the remainder (an arc light is excellent), or the liver
may be removed and cut into slices. In injected specimens the
liver is hopeless.

I washed with alcohol the blood out of the vessels of a fetus
4 inches long and cleared it in wintergeen oil, then injected it
with mercury. This method has the advantage that the extent
of the injection may be watched and controlled.

The injection may be limited by using a coarse granular pig- —
ment that will not go into the capillaries. A gelatine mass is
not absolutely necessary to hold the pigment. A light colored
opaque pigment has the advantage that it may be seen by trans-
mitted or reflected light.

The arteries may be injected and the haemoglobin fixed in the
veins, giving handsome specimens. If it is desired to show only
the injection, no formalin should be used. Much of the haemo-
globin may be dissolved out by putting the fresh specimen into
weak alcohol or alcohol and acetic acid. All of the haemoglobin
may be removed with dilute acetic acid provided an injection
is used that is not affected by this acid.

MATERIAL FOR HISTOLOGY AND EMBRYOLOGY 59

APPENDIX
ON THE ARTERIES AND VEINS IN A 30 MM. PIG EMBRYO

The method of fixing the haemoglobin and clearing in winter-
green oil to show the course of the vessels has been especially
successful in case of pig embryos of about 30 mm. length Figures
2 and 3 show the larger vessels of the median plane and left side
of one of them. The courses of most of the vessels approach
the type of the adult pig and show distinctions in topography
from those in man. The common carotid artery and (right)
innominate artery arise from a common trunk, the brachio-
cephalic artery. The posterior inferior cerebellar artery arises
from the basilar instead of from the vertebral.

Notwithstanding the great development of the vena cava, the
left posterior cardinal is of considerable size. The right cardinal
(not figured) is smaller. The thoraco-epigastric vein is divided
into two parts, one of which drains anteriorly into. the internal
mammary.

The vessels of the limbs could not be completely followed, but
enough was seen to demonstrate that they differ very much from
those in the adult.

Besides the vessels, the mouth cavity, brain, eye, endolymph-
atic labyrinth, lungs, mesonephros, kidney, testis and penis are
outlined in the figures.

McCLENDON

F.

J.

60

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