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SAGE ENDOWMENT FUND

THE GIFT OF

HENRY W. SAGE

1891

Cornell University

The original of this book is in
the Cornell University Library.

There are no known copyright restrictions in
the United States on the use of the text.

http://www.archive.org/details/cu31924024795027

Number 11 OCTOBER, 1920

THE AMERICAN ANATOMICAL
MEMOIRS

NUMBERS I TO 7 INCLUSIVE APPEARED AS MEMOIRS OF
THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY

EDITED BY

GEORGE S. HUNTINGTON
COLUMBIA UNIVERSITY
WITH THE COLLABORATION OF
CHARLES R. STOCKARD AND HERBERT M. EVANS

CORNELL UNIVERSITY MEDICAL SCHOOL UNIVERSITY OF CALIFORNIA, BERKELEY

THE PIGMENTARY, GROWTH AND ENDOCRINE
DISTURBANCES INDUCED IN THE ANURAN
TADPOLE BY THE EARLY ABLATION
OF THE PARS BUCCALIS OF
THE HYPOPHYSIS

P. E. SMITH
ANATOMICAL LABORATORY OF THE UNIVERSITY OF CALIFORNIA

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THE AMERICAN ANATOMICAL MEMOIRS

THE PIGMENTARY, GROWTH AND ENDOCRINE
DISTURBANCES INDUCED IN THE ANURAN
TADPOLE BY THE EARLY ABLATION
OF THE PARS BUCCALIS OF
THE HYPOPHYSIS

PHILIP E. SMITH

ANATOMICAL LABORATORY OF THE UNIVERSITY OF CALIFORNIA

1920

PUBLISHED BY
THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY

PHILADELPHIA
i

on

CONTENTS

. Introduction.......... Secvdieotietaan tee ean e eats de cee Se ayshd eincaneas ephe akdeue tied 5

The conditions of the experiment.............. 0.00 cece eee een eee 7

. Alterations in the pigmentary system.......... 0.0. c cece eee eee 10

The chromatophores of the albinous larvae....................0000 000s 14

The epidermal melanophores...............00 0000 c eee ee ence eees 14

The deep melanophores... ......... 0.0. cece cece ete et nee 18

The xantholeucophores............. 0.000 c cece eee eee eee 18

Hpidemial transplants: < «2x4 a0< 04 deeds daha wewhoweicdmenes ameter eede 23

The effect of various diets upon the pigment cells...................0.. 27
The responses of the chromatophores to various physiological and phar-

macological agents),.:.cosersc¢eay iad eedd Ses daw Hee oa Be eee awe os 30

} Growth: disturbances)....55.4-aveee.accd s.cG eee ho gavage wan dare longed be, Babee aoe 41
The growth rate of the albino; the effect of the administration of various

physiological substances upon this growth rate..................... 44

The growth rate of thyroidectomized larvae..............-.00 000 eevee 67

. Modifications in the size and structure of the endocrine organs............ 68

The hypophysial components.........6... 0.0. c cee ete eee 69

he thyroid ss a:.oeecese ae cee ay beak ae Ones ae Semmes Rearend aka 83

The adrenal cortex and medulla.......... 0... cece cece ees 90

Theepithelial, bodies. sys eis jay dowel warue et ss Ree ceed sae eRe 5 oe 97

Whe faHOR@ aM a esc drat iid cs died oe GO InSeatnat dels SRE E EE Se See ee RR 99

S ADISCUSSIOM aie heal do eth baste tamil’ eangAlinn seed aera Mase Da ohne AUN RA eNO ate 100

2 UMMM AR Yee ha acca SOT ee nae seen Em ae ee ee BES Gee 103

Bibliography: «. ¢sisse2. oewece ak lec ee osieer ase shale eee b oumeere se 109

THE PIGMENTARY, GROWTH AND ENDOCRINE
DISTURBANCES INDUCED IN THE ANURAN
TADPOLE BY THE EARLY ABLATION
OF THE PARS BUCCALIS OF
THE HYPOPHYSIS!

1. INTRODUCTION

Only within the brief period of four years has the favorable
character of the early amphibian tadpole for analyzing the func-
tional nature and the reactions of the members of the endocrine
system been recognized. This may indeed seem strange, since
the value of this material, to which attention was called by
Born and which has been used so advantageously by Har-
rison, in the solution of those problems requiring experimental
procedure upon the early embryo has long been appreciated.
The early amphibian tadpole is peculiarly useful in experimental
biological investigations, since in its earliest stages it is avail-
able for operation and has the inherent capacity to survive
the most severe mutilations. These characteristics make a
special appeal in studies upon the functional interrelationships
obtaining in the endocrine system, for derangements in this
system can be induced by the ablation of certain of its members
in their early embryonal and non-functional stages by a simple
operation, in itself not harmful. The early removal of a gland
will afford then knowledge not only of the essentiality of this
gland per se, but also concerning the dependence of the other
endocrine organs upon this gland for their full development.
This interdependence may be productive of even greater struc-
tural changes in the other glands than a later operation would
produce, because of the lability inherent in embryonal structures.
Further, the utilization of embryonal material would appear

1 Aided by a grant from the Research Board of the University of California.
5

6 PIGMENTARY GROWTH AFTER ABLATION OF

to be necessary in studies upon hypophysial extirpation if the
prolonged survival of the animals is desired, since the complete
extirpation of the epithelial hypophysis? in the adult invariably
proves fatal within a brief period. And it would appear that
only by the prolonged survival of the animal can the maximal
alterations in the other members of this correlative system fully
express themselves, as is shown by the structural picture pre-
sented by the endocrine organs of the ‘hypophysectomized’
tadpole. These organs exhibit structural changes exceeding in
magnitude those obtaining in the hypophysectomized mammal.
Indeed, it is possible to look upon these structural alterations
as the expression of a restored functional balance in the endo-
crine system which has permitted the survival of the animal.
One further advantage is bestowed by the use of embryonic
material, for we may discover a more general influence of the
endocrine organ in question on the manner or rate of develop-
ment of all of the tissues and organs of the body—namely, on
growth—and, as we shall see further on, growth effects are
among the most prominent ones manifested by these disturb-
ances.

In 1912 Gudernatsch reported that frog larvae could be meta-
morphosed by thyroid feeding. This remarkable result stimu-
lated Adler in the same year to utilize the tadpole for the abla-
tion of members of the endocrine system. By what now appears
to be a crude method, Adler, in 1912, burned out the pituitary
from half-grown frog larvae. Although the failures were numer-
ous, the death rate high, and in the surviving successful cases
the injury to the neighboring structures severe, nevertheless,
Adler was able to show that the destruction of the pituitary
impaired the thyroid gland and prevented metamorphosis.
Subsequent and far more successful ablations of the hypophysis
have amply confirmed Adler’s findings.

In 1914 the author attempted to remove the epithelial anlage
of the hypophysis in the early larvae of the newt—D. torosus.
As had been the experience of other investigators, this urodele
material proved unfavorable for early operation. The follow-

2 That portion of the hypophysis arising from the oral ectoderm.

THE PARS BUCCALIS OF THE HYPOPHYSIS 7

ing season unsuccessful attempts to transport living frog eggs
from the Middle West were made. Finally favorable material
was secured from the rather scanty local anuran fauna by col-
lecting the adult pairs of the frog, Rana boylei, at the time of
laying. The larvae of this form proved ideal, wounds healed
rapidly, feeding was vigorous, and growth uniform.

An astonishing result of this early removal of the epithelial
hypophysis was now revealed. Characteristic, silvery-colored
larvae—albinos—in great contrast to their darkly pigmented
normal mates, resulted. Similar results were obtained simul-
taneously by B. M. Allen. In a series of papers the writer,
B. M. Allen, E. R. and M. M. Hoskins, and W. C. Atwell have
further confirmed and sought to analyze this discovery. Allen
has subsequently studied in more detail the changes induced
by an equally early ablation of the thyroid gland. No one,
it would appear, has endeavored to confine his attention to the
altered anatomy and physiology displayed by these pituitary-
free ‘albinos.’ The present monograph aims to do this. While
brief statements of some of its material have appeared from time
to time, there has been collected here for the first time as com-
plete an account as is now possible of the pigmentary upset
and other correlated bodily changes, especially endocrine alter-
ations called forth by this procedure.

The conditions of the experiment

All amphibian material is not equally favorable for work of
this nature. In order to assure prompt healing, the yolk must
be moderate in amount and of a cohesive nature. Thus oper-
ative work upon the larvae of the common newt, D. torosus, is
precluded, due to its large, abundant, and non-cohesive yolk
granules, which extrude for hours through the wound, even
final healing being prevented. The unfavorable character of
the Amblystoma punctatum for work of this nature has pre-
viously been commented upon by Harrison. For studies on
growth it is further essential that the animals be vigorous feeders
and develop at a rather uniform rate. This desideratum would

8 PIGMENTARY GROWTH AFTER ABLATION OF

apparently rule out one of the common California frogs, R.
aurora dratonyii, as well as the newt. The frog, Rana boylei
Baird, and to a limited extent the toad, Bufo boreas, were utilized
in this work and have proved very satisfactory, since they re-
cover quickly from the operation, are vigorous feeders, and
exhibit a rather uniform rate of growth.

The anlage of the epithelial hypophysis can be more easily
ablated than any of the other glands of internal secretion save
the thyroid. As is well known, this ectodermal invagination,
lying between the brain and the pharynx, is connected with the
surface epithelium in very young frog larvae,’ and is conse-
quently readily accessible (fig. 12). If a transverse cut with
finely ground needles be made through the surface ectoderm
between the forebrain protuberance and. the pharynx‘ (figs. 11,
12) and these two structures gently separated, the hypophysial
ingrowth is readily distinguished lying on the ventral surface
of the brain, from which it can be separated without serious
injury to the latter. The stage selected for the operation should
neither be too young, in which case the structures are undiffer-
entiated and serious injury may be done to the mouth, nor too
old, in which case the hypophysis will have migrated to its
deeper position, thus making its removal extremely difficult.
Larvae of 344 to 4 mm. in length, at which time the tail bud is
well formed and the nasal placodes distinct, appear to be in
the most favorable stage for epithelial hypophysectomy (fig. 11).
Since reflex movement has not yet appeared, no anaesthetic is
necessary.

It is essential to furnish a continuously renewed supply of
well-aerated water to the larvae. In this locality the added
necessity of ‘sterilizing’ the water by heat at 60°C. for at least
an hour, followed by cooling and reaeration, has been forced
upon us (Smith, ’18), because of the pathogenic organisms which

3 Excellent descriptions of the development of this structure in Amphibia have
been given by Orr (’89), Corning ('99), Kingsley and Thyng (’04), Atwell (18),
and others.

4A depression at the point of invagination of the hypophysis plainly marks its
position (figs. 11, 12, Hyp. p.). With later development this pit gradually dis-
appears or merges into the stomodaeum. :

THE PARS BUCCALIS OF THE HYPOPHYSIS 9

apparently gain ingress through the city water supply. Experi-
ence during the past three seasons has abundantly justified
this ‘sterilization,’ since infection has been inversely propor-
tional to the extent to which this treated water was utilized.
Such treatment of the water appears to impair in no way the
normality of the animals raised therein.

With the daily feeding of special or check food substances,
as has been done in this study, the necessity of removing the
uneaten particles which would otherwise putrify has forced us to
have special containers manufactured. Our needs were met by
having vessels of two sizes cast. One size, made of a gray glazed
earthenware, is 12 inches square by 6 inches deep; the other
size, made of a gray porcelain, is 6 inches square by 4 inches
deep. The water level in either type can be readily changed
by an L-tube draining through a hole near the bottom of the
container. It was found that from fifty to seventy-five speci-
mens could be reared in each of the larger containers without
overcrowding and from ten to twenty specimens in each of the
smaller ones. The daily cleaning of such containers proved
not to be an onerous task when done with a syphon cleaning
tube fitted with an appropriately shaped inlet.

Various check and special food substances have been fed:
liver, muscle,* anterior lobe, posterior lobe of the hypophysis,
‘adrenal cortex, adrenal medulla, as well as various extractives
and residues of the anterior lobe of the hypophysis. As a check
diet, finely ground fresh liver has proved more satisfactory than
muscle, since the connective tissue of even finely ground muscle
often unites fragments and thus prevents complete deglutition
of partly swallowed series of food particles, resulting in the
death of the animals. In all cases an abundant supply of boiled
lettuce has been furnished. Although liver has proved satis-
factory for the normal tadpole, yet by far the most adequate
food substance for raising a vigorous, healthy albino is the fresh
anterior lobe of the beef hypophysis. With this diet, which

5 Mendel and Osborne (’18) report that the proteins in both muscle and liver
are adequate for the needs of nutrition in growth.

10. PIGMENTARY GROWTH AFTER ABLATION OF

as will be shown replaces the growth substance lost by hypophy-
sectomy, the albinos thrived and were much more healthy,
vigorous, and enjoyed a greater longevity than with any other
diet. This gland proved to be a very desirable food substance
for the normal tadpole as well.

The alterations referable to hypophysectomy in frog or toad
larvae express themselves in several ways. There early appears
a pigmentary disturbance productive of a silvery tadpole—
the albino. This system then exhibits profound structural and
functional modifications. An early retardation in growth is

‘apparent which progressively becomes more marked with devel-

opment. The growth curves of these animals differ not only
in their magnitude, but in their character when compared to
the normal. And finally, most of the other members of the
internal secretory system present a structural picture differing
greatly from the normal.

It is thus convenient to present the matter under three head-
ings:

Disturbances in the pigmentary system.

Alterations in the growth rate.

Alterations in the other glands of internal secretion.

2. ALTERATIONS IN THE PIGMENTARY SYSTEM

The striking color changes which many of the lower verte-
brates exhibit under changed environmental conditions has long
been known and early attracted attention to the mutual inter-
play of the various components of the chromatophore groups
by which this chromatic ‘adaptability’ was effected. In addition
to these environmental color changes, it has also been shown
that pronounced changes in certain of the chromatophore groups
can be experimentally induced: by the injection of adrenalin
into the frog, Lieben (’06); by the immersion of the entire animal
or portions of it in certain endocrine extracts, McCord and
Allen (17), Lowe (17), Spaeth (713 and 718, with fish scales) ;
by experimental operative procedures, Lister (’58), Bimmerman

THE PARS BUCCALIS UF THE HYPOPHYSIS 11

(78), Biedermann (’92), Hooker (12), Redfield (’16); by elec-
trical stimulation, Hermann (’86), Winkler (’10), Spaeth (16,
with fish scales); by pharmacological reagents, Lowe (’17); as
well as by producing numerical and physiological modifications
in the pigment cells by rearing the larvae in different back-
grounds, Babak (’13).6 It is not surprising that a mechanism
exhibiting such a functional lability would be modified by dis-
turbances in the endocrine system; indeed, that this pigmentary
mechanism is influenced by the internal secretory system has
been suggested by Fuchs (’14) and Redfield (16,18). It was not
known, however, until the reports of the writer and of B. M.
Allen appeared in 1916 that the ablation of one of the endocrine
glands would in itself profoundly and permanently modify the
pigmentary system. It was then shown that the early ablation’
of the pars buccalis of the hypophysis induced pigmentary
changes leading to the formation of a ‘silvery’ tadpole—the
albino—the most striking pigmentary alteration as yet effected
in the tadpole. In this pigmentary disturbance all components
of the chromatophore system have been pictured as playing a
significant réle by various writers.

The epidermal melanophores have been shown to be dimin-
ished in number (Smith, Allen), in pigment content (Smith),
and to display a persistent contraction (Allen), all of which
has been substantiated in the recent article by Atwell and by
the author. It has further been shown that the free melanin
which lies near the outer border of the peripheral layer of epi-
dermal cells suffers a pronounced diminution (Smith).

’ By this diminution in the melanin content of the epidermis,
a modification which lends greater transparency to this epithelial
covering, the iridescent quality of the subjacent chromatophore
group—the xantholeucophores—which in the albino display a

6 The literature on the pigment cells is stupendous, only a glimpse of it being
given here. A complete bibliography on this subject will be found in the compre-
hensive article of Fuchs in Wintersteins’ Handb. d. vergl. Physiol., Bd. 3, 1 Hilfte,
2 Teil.

7 An early operation appears to be essential for the production of a pigmentary
effect, since Adler reports no striking pigmentary disturbance subsequent to hy-
pophysectomy in the midlarval stages.

12 PIGMENTARY GROWTH AFTER ABLATION OF

broad and persistent expansion, is permitted full effect. This
striking and persistent expansion in the xantholeucophores or
‘interference’ cells appears to have heretofore escaped attention,
yet in this phenomenon of albinism they play no secondary
role, since to them is referable the silvery and iridescent quality
of the albino.®

The contraction of the deep melanophores, first described by
Allen and later confirmed by Atwell, who indeed refers the
picture of albinism primarily to the altered physiological state
of these cells, the author can corroborate for the young albinous
tadpole, but repeated examinations have failed to reveal a.
definite contraction in the older albinous larvae of R. boylei.
As will be subsequently pointed out, the physiological condition
of these cells could make no significant contribution to the
picture of albinism.

To anticipate, then, what will be more fully shown in this
section, the essential pigmentary changes® contributing to the
picture of albinism in this form are three.in number:

1. A diminution in the epidermal free pigment.

2. A diminution in the number and melanin content of the
epidermal melanophores (because of their paucity in number
and pigment content, the contraction of these cells plays no
important réle in the formation of this picture).

3. A great and persistent expansion in the xantholeucophores.

It is most essential in a study of this nature that the environ-
mental condition be well known on account of the adaptability
of the pigment system to such external factors. The standard
environmental condition, which we believe closely approximates
that obtaining in nature, was furnished by a diffuse light, a gray
background and room temperature (18° to 25°C.). The ex-

8] have recently called attention to the significant contribution which these cells
make to the picture of albinism (Proc. Soc. Exp. Biol. and Med., 43-1418, 1919).

§ Although the pigmentary system of a hypophysectomized tadpole 12 mm. or
even less in length shows variations from the normal, nevertheless, an animal two,
or better, three or more times this size has these modifications more clearly differ-
entiated. The descriptions, except when otherwise stated, were made upon animals
raised upon an anterior-lobe diet and which have attained a size in excess of 45 mm.
and were not less than 314 months old.

THE PARS BUCCALIS OF THE HYPOPHYSIS 13

treme variations from this ‘indifferent’ condition have been
assumed to be a) a white background with direct sunlight;
b) a black background and the absence of light, temperature in
all cases being noted and regulated to suit the experimental
desiderata.

The observations upon the pigment cells have been made
both upon the well-illuminated living animal with the binocular
microscope (a Zeiss instrument fitted with a water-immersion
objective and no. 2 oculars; magnification, 42 diameters) and
by examination of cutaneous whole mounts. By the first
method the progressive changes in the pigment cells under
altered environmental conditions can be noted, the observations
in most cases of necessity being rapidly taken so as to exclude
possible alterations resulting from the rather brilliant illumina-
tion necessary for binocular observation (as, for instance, in
observing a dark-adapted animal). These observations have
been checked and supplemented by skin whole mounts of the
fixed animal, the fixation being so rapidly effected as to preclude
any physiological alterations which might take place. Observa-
tions have been largely limited to the dorsal and neighboring
lateral portions of the body, save in certain cases where the
pigment cells lining the body cavity were noted in fixed speci-
mens. Although observation of the deep melanophores of the
dorsal region involves some difficulty more especially in the
albino, in which they are largely masked by the expanded xan-
tholeucophores, yet the observations herein reported were re-
stricted to this region, since repeated observations have revealed
the fact that the pigment cells exhibit regional variations both
in the time and the magnitude of their response.

In this section of the paper there will be presented:

1. The anatomical and physiological characteristics of the
pigment cells of the albino as compared with those of the un-
operated tadpole and some remarks on the development of this
condition.

2. The responsibility of the endocrine system for this pig-
ment fault as shown by—

14 PIGMENTARY GROWTH AFTER ABLATION OF

a. Epidermal transplants.

b. The effect of various pabula upon the pigment cells.

c. The response of the chromatophores to various physio-
logical and pharmacological agents.

The chromatophores of albvnous larvae

As is well known, the melanophores of the tadpole are of two
types. One type—the epidermal melanophore—is found in the
epithelial covering of the body; the other type—the deep melan-
ophore—lies in or around the deeper structures. These two
types, then, of necessity, will be treated independently.

The epidermal melanophores. The epidermal melanophore,
when in an expanded condition, presents an irregularly shaped
body from which branched, slender processes radiate for rela-
tively long distances (fig. 13). When in a greatly contracted
condition, these processes are not evident and the cell body
then is of a spherical or slightly irregular shape (figs. 19 and 21).
All intermediate conditions between these two extremes can
be seen with proper light and temperature conditions.

The melanophores of the epidermis are greatly reduced in
number in the albino (figs. 13 and 14), many counts showing
an average reduction of two-thirds from the normal number.
Even in a greatly expanded condition these cells are usually
separated by wide intervals, a condition which contrasts sharply
with that obtaining in the normal animal where the expanded
processes unite with, or even overlap, each other. Thirty-one
counts from five specimens gave an average of 38 epidermal
melanophores in a unit area of 0.36 of asq.mm. Similar counts
from normal animals reveal 119 to this unit area, thus exceeding
by over three times the number present in the albino (table 1).
It is emphasized, however, that the distribution of these cells
in neither type ef animal is uniform. Yet their number in the
normal even in the areas where they are most thinly distributed
exceeds very considerably their number in the albino where
they are most thickly placed. Further, the actual melanin
content of these cells is diminished in the albino (figs. 13 and 20).
Their processes, slender and of light color, present a very differ-

THE PARS BUCCALIS OF THE HYPOPHYSIS 15

ent appearance from that furnished by the course black processes
of the normal melanophore.'?

Not only do these superficial melanin-bearing cells exhibit
differences of a quantitative nature in these two types of ani-
mals, but constant physiological dissimilarity is also encountered.
It may be urged that the functional states of these cells in the
normal usually exhibit such wide variation not only between
two or more individuals, but also in the cells of a single indi-
vidual, that the determination of a normal physiological con-
dition is precluded. Certain it is that not only do we find
differences between the condition of the individual cells of a
specimen, but under identical environmental conditions these
cells in one animal may be expanded while those of its mates
are partly contracted.1 Yet this variability is not so great as
to preclude our defining the condition of these cells in a normal
animal in an ‘indifferent’ environment as one of complete or
nearly complete expansion (fig. 13).

With the albino, as with the normal, but to a lesser extent,
the same variability in the superficial melanophores of various
individuals is exhibited. The individual cells of any one speci-
men, however, usually exhibit a greater dissimilarity than in
the normal animal. <A very few of these cells are almost in-
variably broadly expanded, a few are half expanded, while the
majority are completely contracted or but slightly expanded
(fig. 14). It is apparent, then, that the usual physiological

10 The evidence on this point is quite clear; corroborative evidence has also re-
cently been furnished by Atwell, although Allen is apparently not in agreement
with these findings, for he says: ‘These observations and a careful study of the
pigment cells convince me that there is no disappearance and bleaching of the pig-
ment cells as asserted by Smith.’”’ It is worthy of mention here that the author has
never mentioned a “disappearance and bleaching of the pigment cells,” stating,
“Counts of the epidermal melanophores—in the albinos and checks show that the
number of these cells in the epidermis is reduced in the former’? and—“ the melano-
phores of the albino specimens contain fewer pigment granules than do those of
the checks and thus have a distinctly lighter appearance.” The process leading
to this condition was not then discussed by the author.

11 This variability has led certain investigators to confine their observations in

various physiological experiments to the more uniformly reacting deep melano-
phores—for example, Laurens.

16

PIGMENTARY GROWTH AFTER ABLATION OF

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18 PIGMENTARY GROWTH AFTER ABLATION OF

condition obtaining in these cells in the albino is one of com-
plete, or nearly complete, contraction.

The free pigment which forms a definite, though imperfect,
layer in the normal animal is much diminished in amount and
is irregularly distributed in the albinous larvae (figs. 13, 14).
In the skin whole mounts of the albino, it indeed forms but a
scanty and irregular sheet which stands in contrast to the very
distinct layer present in the normal animal.

The deep melanophores. The number and melanin content
of the deep melanophores apparently are not altered by the
ablation of the hypophysis. This structural independence ap-
pears not to lend support to those investigators who assert that
both groups of melanophores, epidermal and corial, arise from
a common source.”

That the deep melanophores exhibit a contracted condition
has been noted by Allen and Atwell, the latter, indeed, referring
the phenomenon of albinism primarily to their contracted con-
dition. Many examinations of larvae in excess of 45 mm. in
length, made both on the living animal and after rapid fixation,
convince the author beyond question that in the body and head
regions these cells, whether occurring in the corium, in muscle
sheaths, or in the fascia of the viscera and body cavities, exhibit
nearly the same physiological state in the fully developed albino
of R. boylei as in the normal (figs. 13, 14); in the young albino,
however, certain of these cells appear to be in various stages of
contraction (fig. 16).

The xantholeucophores. We have noted significant and con-
stant changes in the superficial layer of the first group of pig-
ment cells—the bearers of melanin. Changes no less significant
in the phenomenon of albinism are encountered in the second
group of chromatophores, the bearers of guanine and xanthine.
Indeed, since the silvery and iridescent characteristics of many
fishes are referable to these cells, it would seem to make them of
paramount importance in the phenomenon of albinism. When
viewed by transmitted light, these cells are of a grayish or brown-

2 Ehrmann (’96 and earlier) refers the common origin of the melanophores to
the mesoblast of the dorsal cephalic region of the body.

THE PARS BUCCALIS OF THE HYPOPHYSIS 19

ish color. It is, however, with reflected light that their great
beauty and their contribution to the color of the animal becomes
apparent. For one constituent, the guanine, distributed in
minute crystals reflects the light and lends to it the iridescent
qualities; the other constituent, the xanthine, small in amount
in the tadpole, contributes to this color effect.

Delicate in structure and apparently exhibiting no resistant
cell membrane, these cells are prone to disintegrate, consequently
appropriate and rapid fixation is essential for their preservation.
This delicacy is well shown in the epidermal transplants where
although but a few seconds may lapse before the graft ‘takes,’
yet these cells in any region where an intimate union between
host and graft does not obtain, change their character and ulti-
mately disintegrate. Or, indeed, even when not subject to
mechanical disturbances, as following the natural death of their
host, they are wont to lose quickly their cellular integrity, form-
ing a diffuse layer of fine crystals, which exhibit a vigorous
Brownian movement—a most striking picture with the polar-
iscope. Not only is early and rapid fixation essential, but
appropriate fixers must be employed. This is well shown if
a specimen be dropped into formol. Such an animal after a
few weeks, or even days, loses its silvery appearance and as-
sumes a gray tone. Microscopic examination reveals the fact
that the xantholeucophores are no longer visible.

Over the dorsal body areas the xantholeucophores form two
well-defined layers, each layer being composed of discrete inde-
pendent cells. The peripheral layer, lying just beneath the
epidermal basement membrane, is usually separated by a con-
siderable interval from the deeper layer which lies adjacent to
but external to the corial melanophores. In those areas where
the epidermis closely approximates the underlying firm body
structures, these two layers of xantholeucophores, except under
high magnification, may appear as a common layer. We thus
see that the iridescent cells lie between the two types of melan-
ophores, an arrangement the recognition of which is of no little
moment in evaluating the réle of the various groups of pigment
cells in albinism,

20 PIGMENTARY GROWTH AFTER ABLATION OF

Both layers of corial xantholeucophores are broadly expanded
in the albino (figs. 14, 16, 18, 20 to 23, 55 to 58), an expansion
which is singularly unamenable, more especially in the older
albinos, to altered environmental conditions. To the broad
expanse of the refractive cells the albino owes its metallic tone, as
can be clearly shown by all procedures which contract these cells.

We have thus far confined our attention to two types of tad-
poles, that type in which the tadpoles have suffered the entire
loss of the buccal component of the pituitary, the albino, and
that type whose. members have suffered no operative inter-
ference, the normal. It is now desirable to consider a third
type—those in which the epithelial hypophysis was incompletely
ablated, the ‘partial’ albino (figs. 52 to 54). This type of tad-
pole usually suffers a serious pigmentary disturbance, its pig-
mentary system simulating that of the albino more than that
of the normal. In fact, its almost typical albinism frequently
makes this animal difficult and often impossible to distinguish
during early development from its completely hypophysectom-
ized brothers. Later in life it is readily distinguishable, since
legs develop in the partially but not in the completely hypophy-
sectomized tadpole. In the albinous type of partially hypo-
physectomized tadpole as in the typical albino the free pigment
is greatly diminished in amount and the epidermal melanophores
are scanty in number. That the epidermal melanophores are
slightly greater in number than in the complete albino, however,
is apparent from counts. It will be recalled that thirty-one
counts from five albinos gave an average of thirty-eight epi-
dermal melanophores to a unit area 0.36 of a sq.mm. Seventeen
similar counts from three ‘partial’ albinos gave an average of
fifty to this unit area (table 1). The xantholeucophores appear
to be no way different from those of the typical albino. They
exhibit a broad and persistent expansion. Although the pig-
ment system of this animal closely resembles that of the albino,
yet, in contrast to the albino, it does not suffer serious structural
defects in any of its endocrine organs save one, the neural com-
ponent of the pituitary. Exhibiting, then, characteristics of
both the albino and the normal, this ‘hybrid’ tadpole has been

THE PARS BUCCALIS OF THE HYPOPHYSIS 21

of especial value in freeing any of the other endocrine glands
which have been studied from responsibility for the pigmentary
disturbance, as will be later shown.

An examination of the pigmentary system of the albino has
revealed that one group of chromatophores, the xantholeuco-
phores, exhibits a great expansion; another group, the epidermal
melanophores, is diminished both in number and pigment con-
tent and displays an unusual contraction. It is necessary, then,
to examine the developmental stages leading to the production
of the characteristic albino if we wish to determine whether
this is a progressive phenomenon; whether from the beginning
the formation of the epidermal melanophores, for example, is
inhibited by the endocrine fault, or whether on the other hand
they form only to partially disappear later. The three groups
of pigment-bearing cells make their appearance in the following
sequence: deep melanophores, xantholeucophores, epidermal
melanophores. <A period of several days intervenes between the
appearance of each of these groups, and so they may well be
treated independently in a developmental study.

The time of appearance of the deep melanophores apparently
is identical in both the normal and hypophysectomized tadpole.
In a 6 to 7 mm. stage for the normal, they have already made
their appearance in the dorsal regions of the head and body.
A few show the typical shape of the deep melanophore. A
majority, however, have longer and more delicate processes than
the typical corial melanophore. They either lie well separated
from the epidermis or, especially where the epithelium closely
approximates the underlying firm structures, may be in contact
with the epithelial covering. From this stage they progressively
increase in number and gradually become organized until they
attain the typical arrangement as a corial sheet at a 10 to 12 mm.
stage (fourteen to twenty days after the operative stage).

The xantholeucophores make their appearance in larvae of
10 to 11 mm. (fourteen to sixteen days after the operative stage),
appearing concomitantly both in time and number in the normal
and hypophysectomized larvae. Slightly more numerous, pos-

99 PIGMENTARY GROWTH AFTER ABLATION OF

sibly, in the hypophysis-free specimens, this numerical difference
is so slight as to be well within the limits of variability. From
their earliest appearance they are expanded in the operated
tadpole (fig. 16) in contrast to their punctate character in the
normal (fig. 15). The early, almost imperceptible albinous
appearance which denotes a successful hypophysis extirpation is
due largely to the expanded condition of these cells, since in
these early stages the free epidermal pigment in both albinous
and normal larvae is identical and the epidermal melanophores
have not as yet formed in either type of specimen. Scarcely
perceptible at first, this whiteness of the hypophysectomized
tadpoles progressively increases with the increase in the number
of the xantholeucophores, and is further emphasized by the
reorganization and diminution in the early embryonic pigment
which takes place, as will be described later.

In the diminution in the epidermal melanin we find the first
structural pigmentary alteration referable to hypophysectomy.
Here we have to deal with the double expression of the melanin,
that existing as free granules and that included in the chromato-
phores. In the earliest stages free pigment is found diffusely
scattered throughout the two cell layers of the epidermis, al-
though occurring more plentifully in the outer of these layers
and in certain cells of this outer layer. At a 10 mm. stage it
has become localized in the superficial layer of the epithelium,
and by the time the larvae have reached approximately a 20
to 25 mm. stage it is found only in the peripheral zone of this
cell layer.* Identical in amount in the earlier larval stage of
the operated and normal larvae (figs. 15, 16), it gradually be-
comes diminished in the albino (figs. 17, 18), a diminution which
aids in permitting the iridescent qualities of the xantholeuco-
phores to be fully expressed and which is shown by the rapid
development of the albinous picture at this time (figs. 42 to 45).

The first typical epidermal melanophores make their appear-
ance in about a 10mm. normal (twenty-two days after the
operative stage, fig. 15). At first scatteringly distributed and

13 The changes here described are nearly identical with those described by Maurer
(’95).

THE PARS BUCCALIS OF THE HYPOPHYSIS 23

light in color, these cells rapidly increase in number and melanin
content; by a 15 mm. stage they have numerically and struc-
turally attained the mature larval condition (fig. 17, table 1).
Their appearance in the albino is more tardy. They do not
appear as early and are always diminished in number and melanin
content as shown by figures 16 and 18, which were drawn from
albinos of the same age and size as the normals from which
figures 15 and 17, respectively, were drawn. A partly con-
tracted condition is also evident from their earliest appearance.

No evidence has accrued from these studies which suggests
that the diminution in the number of epidermal melanophores
suffered by the albino is affected by a migration of the epidermal
melanophores into the corium as suggested by Allen. On the
other hand, the evidence all points to the developmental and
functional independence of the two systems of melanophores.
The first melanophores to appear are those in the corium, and
there is at no time any evidence that their number is augmented
by a migratory process from the epidermis. The formation of
the epidermal melanophores from the beginning is apparently
partially inhibited in the albino, an inhibition which is expressed
not only in the diminished number of cells, but in their melanin
content as well.

One of the remarkable features in this pigmentary disturbance
is its early appearance. Whether referable to some missing
hormone of the anterior lobe or to other hormonal disturbances
provoked by the operation, it appears clear that hormones are
already produced by the small groups of embryonic cells con-
stituting the endocrine glands at a time when the latter exhibit
but little if any of the structural differentiations which char-
acterize the adult internal secretory organs.

Epidermal transplants

We have detailed at some length the pigmentary disturbances
which result from the early loss of the epithelial hypophysis in
the tadpole. It is now of interest to inquire whether this func-
tional upset is due directly to an alteration in the quality of

24 PIGMENTARY GROWTH AFTER ABLATION OF

the fluids bathing these cells or, on the other hand, to alterations
in their nervous mechanism.

Unimpeachable evidence has been secured on this point by
reciprocal skin transplants. In a very considerable number of
cases skin interchanges have been successfully effected between
a normal tadpole and its albinous mate. Invariably, and in a
period not exceeding four hours for the xantholeucophores and a
somewhat longer period for the epidermal melanophores," these cells
of the transplant assume the state characteristic for the corresponding
cells of the host.

The technical manipulations involved in performing these skin
exchanges even in larvae exceeding 40 mm. in length are not
difficult. A narcosis sufficiently deep for operative work has not
readily been effected at room temperature, although serving
well for ordinary examinations in which no trauma was inflicted.
Of the several anaesthetics employed, ethyl eurethane (Merk)
in a strength of 0.5 per cent has proved the most efficacious,
especially when used at a low temperature (1° to 3°). The
lighter narcosis effective at this low temperature is of no
little significance, since not only is recovery more certain, but
also functional changes in the pigment cells inherent in a pro-
found anaesthesia are avoided.

After a sufficiently deep insensibility had been produced, the
specimens were placed closely together in depressions in the
wax plate lining the dish. Incisions on the dorsal epidermis
mapping out an area approximately 46 mm. were then made
on the dorsum of each specimen with the aid of the binocular.
Following this the skin was rapidly freed and the interchange
effected, the grafts being gently pressed down and not infre-
quently tucked under the edge of the host’s skin. These grafts
quickly adhered, and in a short time no movement of the host
could dislodge them.

4 This reaction of the epidermal melanophores of the albinous graft to the normal
host—one of expansion—applies only to the cells originally belonging to the graft.
Epidermal melanophores from the surrounding normally pigmented epithelium
of the host quickly invade the albinous graft. These cells display a state of great
contraction for at least a considerable interval.

THE PARS BUCCALIS OF THE HYPOPHYSIS 25

There now was exhibited one of the most striking phenomena
presented in this work. The heretofore broadly expanded
xantholeucophores of the albinous graft gradually contracted
under the influence of its normal host. In half an hour they
were much reduced; in two hours they were punctate, even
exceeding in minuteness under this new stimulus the diminutive
size of the host’s ‘interference’ cells (figs. 57 and 58). Likewise
the epidermal melanophores of the albinous graft gradually
expanded. This change from the altered physiological state of
these cells in the albino to the state characteristic of the normal
animal then persisted for the life of the host.

A change as striking was exhibited by the heretofore con-
tracted xantholeucophores and expanded epidermal melano-
phores of the normal graft. In about the same interval the
xantholeucophores of the normal graft became broadly expanded
(figs. 57, 58), and after a somewhat longer interval the epidermal
melanophores assumed a punctate condition. These cells, then,
in this brief interval had changed from the functional state they
had so long experienced to the opposite condition exhibited by
them in the new host.

There is, however, the possibility that this remarkable trans-
formation was due to the mechanical manipulation and not to
the influence of the host. This possibility can definitely be
excluded by the simple test involved in an autoplastic skin
graft. Identical incisions were made, the freed epidermis, how-
ever, instead of being transferred to a new host, was returned to
the position from which it for a brief interval had been removed.
In neither the normal nor albinous larvae did these cells suffer
any permanent change, but remained in the same condition
as the cells which had not been removed.

Definite evidence has thus been secured which shows that
the altered state of these cells in the albino has been brought
about by the action of some substance present in the tissue
fluids and not by alterations in the nervous mechanism, since
we can conceive of no reestablishment of nervous connection in
such a brief interval. Further, it would seem probable that this
substance acts directly upon the pigment cell itself and not upon

26 PIGMENTARY GROWTH AFTER ABLATION OF

the severed nerve termination. This would appear to be the
case, since if this atypical condition exhibited by the cells of
the normal graft to the albinous host was due to the action of
the tissue fluids upon the severed motor connections, we would
expect a change in the size of these cells in that interval which
must occur between the degeneration of this nerve ending and
the reestablishment of a new nervous connection, a change which
apparently does not occur. Because of the known potency of
minute amounts of endocrine secretions, the most natural as-
sumption is that this alteration has been effected through some
‘hormonal’ component of the plasma or lymph.

Rather interesting results have been secured by submitting
these operated animals to adrenalin and to ‘light and heat’
stimuli. These experiments, although not as complete as could
be wished, have been carried out on some half-dozen albinous
and normal tadpoles into which grafts from the opposite type
of animal had been made. The response or failure to respond
in the case of host and transplant cells has been nearly identical.
The tests were carried out from twelve hours to three days
subsequent to the operation and supply additional evidence
that the effective force causing a response of these cells to stimuli
is the direct application of the internal secretory substance to
the cell bodies and is not effected through the motor sympa-
thetic endings, since it is probable that any nerve ending severed
as it is from the nerve fiber would have degenerated in this
interval, and even after this lapse of time new connections could
hardly have been established.

Some rather significant evidence has been secured on the
formation and migration of the epidermal melanophores in the
transplants. There invariably occurred an early migration of
the epidermal melanophores from the normal host into or over
the albinous transplant. In a period of a few hours the sulcus
intervening between the two types of skin would become filled
with a dense mass of melanin-rich cells in which the pigmented
epidermal cells could not readily be distinguished from the true
melanophores. In twelve hours, or even less, growing points
would appear from this confused scar tissue which invaded or

THE PARS BUCCALIS OF THE HYPOPHYSIS 27

became incorporated gradually into the albinous graft. In this
invading tissue, and indeed in those parts of the albinous graft
which clearly had not suffered an invasion by the epidermis of
the host, epidermal melanophores could readily be distinguished
which curiously displayed, for a considerable interval, a state
of great contraction. In the end the albinous transplant was
as abundantly supplied with melanophores as was the normal
epidermis. Although the free pigment of the albinous graft
increased more slowly than did the epidermal melanophores,
yet this too ultimately exhibited a normal condition. Thus,
after an interval not exceeding two months, both the free pig-
ment and the epidermal melanophores of the albinous trans-
plant had assumed the characteristics typical of the epithelium
of the normal host. ;

The sequence of changes in a dark graft to an albinous host
is of a quite different nature from the alternate just described.
Although there appeared to be a slight reduction in both the
amount of the free pigment and the number of the melanophores
of the epidermis, yet these changes were of a minor nature. No
extensive migration of the melanophores from the normal trans-
plant where they were thickly congregated into the sparsely
supplied surrounding albinous epithelium occurred, nor by any
other process did any great reduction in the number or pigment
content of the transplanted melanophores take place during
the interval that the animals were under observation (two
months). These cells, once formed, appear to retain their
integrity. This would appear to be the correct interpretation
of the non-effect upon the pigment cells of hypophysectomy in
the midlarval stages (Adler).

The effect of various diets upon the pigment cells

We have just shown by the skin exchanges that the atypical”
physiological state of the xantholeucophores and epidermal
melanophores of the albino is directly referable to an alteration
in the tissue juices which in turn is probably of an ‘hormonal’
nature. If, then, this endocrine disturbance which is inaugu-

28 PIGMENTARY GROWTH AFTER ABLATION OF

rated by an hypophysial deficiency leads to an upset in the pig-
mentary system, will it not be possible with the proper endo-
crine diets to abort or ameliorate this derangement? The possi-
bility appears the more probable since the general growth effects
inherent in the loss of the buccal hypophysis can be prevented
by the administration of the beef glandular lobe, as will be shown
later.

Of the endocrine substances which have been fed, glandular
lobe and neural lobe (with the pars intermedia) of the hypophysis,
adrenal cortex, adrenal medulla, and liver, one only—the neural,
or posterior lobe—has caused a formation of epidermal melanin
approaching the normal, while none have reduced the xantholeu-
cophores to a normal state of contraction.

It was early seen that those specimens receiving the posterior
lobe of the pituitary exhibited a darker appearance than the
albinous larvae fed upon the other fresh glands (figs. 46 to 51).
Although expressed feebly at first, this depth of color became
more pronounced as the feeding and growth progressed. So
pronounced did it become after five months of this diet that
there would have been some uncertainty in identifying these
as hypophysectomized specimens save for the fact that legs
did not develop. An examination of the living animal with the
binocular reveals that although the xantholeucophores are
typical of albinos, i.e., are fully expanded, there has been a great
replacement of the epidermal melanin. This replacement was
readily confirmed by a study of whole mounts of the epidermis
which reveal that both components of the epidermal melanin
have been increased (compare figs. 21 to 23 with figs. 14, 20).
The free pigment is not, as in the typical albino, distributed in
diffuse aggregations, but is found in considerable amounts in
all of the superficial epidermal cells. The epidermal melano-

. Phores also show a depth of color never attained in the typical
albino, as is most clearly shown if the processes of the broadly
expanded melanophores (a condition readily obtained by the
death of the animal) of the posterior-lobe-fed albino be com-
pared with those of the albinos on any other diet.

THE PARS BUCCALIS OF THE HYPOPHYSIS 29

Not only are the melanophores darker in the albinos receiving
posterior lobe, but the number of these cells is also increased
(table 1). Instead of averaging but thirty-eight to the unit
area, they average in excess of fifty to this unit area (0.36 of a
sq.mm.). Thus both in number and pigment content the epi-
dermal melanophores of these animals are intermediate between
those of the normal specimens and the typical albinos.'5

It is not on the structural side alone that we find changes
with a posterior lobe diet. The epidermal melanophores of
these posterior-lobe-fed specimens uniformly exhibit a great and
persistent contraction; they are uniformly either rounded or,
at most, slightly irregular in shape, appearing as intense dark
dots in all areas of the dorsal epidermis (fig. 21). Moreover,
this contraction is persistent. After days of the substitution of
another diet, it still persists; indeed, under the most potent
stimulus—light and heat—for causing an expansion of the
epidermal melanophores of the albino, a considerable interval
must intervene in which neural lobe is not fed in order to obtain
a decisive expansion of these cells.

It is curious that no increment in the epidermal melanin was
produced in the unoperated tadpoles with a diet of fresh posterior
lobe. Not only did no pigmentary increment result, but the
contraction of the epidermal melanophores so typical of albinos
on this diet did not regularly occur, or at most was but transitory.

Of the other substances which have been fed, none have ap-
preciably affected the epidermal melanophores. One, namely,
liver, has produced unmistakable differences, however, in the
appearance of the xantholeucophores as contrasted to the albinos
upon other diets. This effect is not expressed in any alteration
in the usual extreme expansion of these cells, but rather in their
color. These specimens do not show the golden sheen of the
anterior or posterior lobe or adrenal cortex fed animals, but
exhibit a chalky whiteness especially noticeable in the gill regions
(figs. 46, 48), an appearance probably due to a reduction in the
lipochrome content of the cells.

16 The author has not supplied albinous larvae with posterior-lobe extracts. It
is apparent from Allen’s work, however, that if they are potent in causing a melanin
increase, they must be fed for a considerable interval of time.

30 PIGMENTARY GROWTH AFTER ABLATION OF

The responses of the chromatophores to various physiological and
pharmacological agents

It has been shown that the pigment cells of both the normal
and the albinous tadpole are influenced by the internal secretory
system. Since the endocrine system is abnormal in the albino,
it is logical to inquire whether the atypical pigmentary system
of these specimens will react to various stimuli as does this sys-
tem in the normal animal.

Although we can largely confirm Laurens’ observations (made
on Amblystoma) upon the irregular reactions of the epidermal
melanophores, it will be recalled that in the unoperated tadpole
the usual state of these cells under indifferent environmental
conditions—diffuse light and a gray background—is one of
two-thirds to full expansion. The xantholeucophores, on the
contrary, are punctate or slightly expanded, while the deep
melanophores are fully expanded.

The usual condition of the epidermal melanophores of an
albino in our so-called ‘indifferent’ environment was one of
complete, or nearly complete, contraction. The xantholeuco-
phores, on the contrary, are broadly expanded.

If we now place a large albinous and a large normal’ specimen
in the dark for a period of one to two hours at room temperature,
we find certain well-marked changes take place. The epidermal
melanophores of the normal, if not completely expanded, become
so, but those of the albino being previously greatly contracted
are unaffected. The xantholeucophores of the normal become
punctate; these cells in the albino remain fully expanded. The
deep melanophores of both animals become greatly contracted,
the animals then displaying a ‘transparency’ typical of the con-
tracted condition of these cells. A low temperature (0° to 15°)
apparently does not increase the reaction to any extent, the
epidermal melanophores of the albino surely not being affected.

16 As complete an analysis of the reactions of the epidermal melanophores in the
young normal tadpole as could be desired has not been made. The reactions here
described unless otherwise stated apply only to large larvae at a time shortly pre-
ceding metamorphosis (in the normal).

THE PARS BUCCALIS OF THE HYPOPHYSIS 31

If we now subject these large animals to the opposite environ-

. mental conditions (direct sunlight, a white background, and a
high temperature—33° to 35°), we see a striking change take
place. The epidermal melanophores of the normal frequently
gradually contract and may ultimately after an hour or even
less at times assume only a slightly expanded condition (fig. 19).
These cells in the albino, on the contrary, invariably expand,
ultimately exhibiting a fully expanded condition (fig. 20). There
is thus frequently exhibited a reversal in the reaction of these
cells in the large specimens of these two types of animals,’ a
reversal not evinced to its fullest extent with any stimulus tried
by the author save that of heat combined with direct sunlight.
Either extreme heat or light evokes this reaction to some extent,
the relative importance of each factor, however, being difficult
to determine. A normal specimen, if placed in the dark at
30° to 35°, may exhibit after two to three hours partly contracted
superficial melanophores. If the temperature becomes normal,
these cells expand; if the temperature is maintained and sun-
light used, further contraction not infrequently takes place.
An albino, if placed in the dark at 30° to 35°, exhibits in two

1 One of the most striking features of the experimental work upon the epidermal
pigment cells of the albinous and normal larvae has been the invariable response
of these cells in the former as contrasted to their uncertain response in the latter.
Of the very considerable number of albinous larvae which have been subjected to
the stimulus of ‘light and heat’ a response, one of expansion, has invariably been
exhibited by the epidermal melanophores. In contrast to this stands the variable
response of these cells in the normal larvae. Because of the influence which the
internal secretory glands have upon these pigment cells, this invariable response
in the albino would appear to be of considerable significance, since certain of the
endocrine glands in this type are structurally and also presumably functionally
deficient. Consequently, we could expect that the response of these cells in this
form would be more certain than in the normal where these cells are under the
variable influence of the internal secretory products.

In 1919 (Proc. Soc. Exp. Biol. and Med., 44-1419) I called attention to the op-
posed reaction of the epidermal melanophores in the albinous and normal larvae.
The experimental work upon which this description was based was carried out
upon the older albinous and normal larvae and should have been so stated. These
normal larvae very uniformly exhibited a contraction of their epidermal melano-
phores when subjected to ‘heat and light’ as contrasted to the expansion of these
cells in the albino under identical conditions. Subsequent work has revealed a
more variable response of these cells in the normal, but not in the albino.

32 PIGMENTARY GROWTH AFTER ABLATION OF

hours partly expanded epidermal melanophores; if the tempera-
ture becomes normal, they contract to the usual condition; if
the temperature is maintained and sunlight used in addition,
they expand to the maximum. Thus we see that, although
high temperature alone evokes the reaction, it is incomplete
and slowly expressed, double the time being necessary. The
same condition prevails if light alone is used. The reactions are
evoked slowly and incompletely. Thus a combination of both
light and heat is necessary to evoke the maximal response in the
shortest time. ;

It has been mentioned that the xantholeucophores of the
normal are uniformly nearly punctate in a dark-adapted animal.
If the large normal ‘animal be subjected to high temperature
and direct sunlight, these cells become broadly expanded (fig.
19), never exhibiting, however, as great an expansion as invari-
ably is presented by these cells in the albino. Thus we see that
in the large normal animal there is frequently a reversal in the
reaction between the ‘interference’ cells and the epidermal
melanophores—one expands and the other contracts. Such a
correlated opposite reaction of the pigment-cell groups no longer
holds in the case of the albino, inasmuch as the xantholeuco-
phores are always maximally expanded there. However, since
under the usual environment the epidermal melanophores of
the albino are almost maximally contracted, these two groups
of cells are consequently usually in an opposite physiological
condition, as is the case in the large normal larvae. We have
already given evidence for our belief that the failure of albino
xantholeucophores to react to stimuli is due to a tonic hormonal
disturbance present in the tissue juices.

The deep melanophores with a white background and light
expand; in darkness they contract; thus, in both types of ani-
mals their reaction is identical under all the environmental
conditions employed by the author.

Specimens that have been thyroidectomized at an early stage,
as is well known, exhibit no pigment deficiencies. Under the
conditions of light and heat or of darkness, they react as do
their unoperated brothers, with this difference: the reaction,

THE PARS BUCCALIS OF THE HYPOPHYSIS 33

although of an identical nature, is more slowly evoked. In
determining the possible cause for this tardy response, the work
of Levy is particularly suggestive. This investigator has shown
that the reponses to adrenalin are more rapid if the thyroid
has been stimulated or its extract injected. Although it has
not been shown to what extent these responses of the pigment
cells are referable either to the adrenal or directly to the sym-
pathetic nervous system, still the slower responses of thyroidless
larvae may well be of considerable significance in view of Levy’s
work. Furthermore, since pigment cells are formed and react
in the thyroidless specimens in an identical manner with those
of the normal, it is apparent that neither their atypical forma-
tion nor reaction in the albino can be referred to the atrophy
of the thyroid effected by the hypophysectomy.

It has been shown that the epidermal melanophores of the

- posterior-lobe-fed hypophysectomized specimens exhibit a uni-
form state of great contraction, a contraction that persists for
an extended period after the substitution of some other diet.
Will these cells then respond to light and heat as do those of
the typical albino?

It will be recalled that in the typical albino these cells, unlike
the xantholeucophores, are sensitive to brilliant illumination
coincident with increased temperature, undergoing an expansion.
But the epidermal melanophores of the posterior-lobe-fed albinos
stubbornly maintain their contracted condition with this stimu-
lus and appear incapable of responding. Lest it be supposed
that the reaction of these cells has approached that frequently
exhibited by normal larvae, one need only to try their response
to complete darkness where normal behavior would give us full
expansion. Under all circumstances, their contracted condition
is maintained just as is the corresponding persistent expansion
of the xantholeucophores in these hypophysectomized larvae.

It was highly interesting to note that an abrupt change in
the diet of the posterior-lobe-fed albinos, a substitution of a
liver diet, led gradually to a resumption of the response char-
acteristic of the typical albino. After an interval of one week

in which they have received no nervous hypophysis they become
MEMOIR NO. 11,

34 PIGMENTARY GROWTH AFTER ABLATION OF

definitely expanded (fig. 22) when subjected to heat and direct
sunlight. This response becomes more and more marked, until
after five weeks we have an almost typical response (fig. 23).
The xantholeucophores in these animals as in the typical albino
remain broadly expanded under all environmental conditions.
Posterior lobe feeding, then, which gives us an epidermal melano-
phore system which approaches the normal in melanin content,
does not permit these cells to exhibit normal responses to light
and heat. It merely maintains them in a refractory and con-
tracted state, and when the feeding is stopped they exhibit the
typical physiology of the albino.

The stimuli to which the two types of larvae have thus far
been subjected are such as they might meet in the extremes of
their normal environment. There is yet another class of stimuli
which may be employed, namely, pharmacological agents, in
which the larvae may be directly immersed. By this means we
may be able to secure further evidence in regard to the endocrine
locus responsible for the peculiar functional state of the chroma-
tophore groups in the albino. Of the substances employed
(pars intermedia emulsion, pituitrin, adrenalin, pineal gland
emulsion), the first—pars intermedia emulsion'*—from the pro-
found pigmentary changes which it induces in the albino, es-
pecially merits attention. Atwell has called attention to the
darkening of albinous larvae when placed in emulsions or ex-
tracts of this substance and has noted the response—one of
expansion—of the deep melanophores to this treatment, a re-
sponse which led him to refer the usual picture of albinism to
a contraction of this melanophore group, and conversely the
darkening of these treated albinous larvae to the expansion of
these cells. We can fully confirm Atwell’s observations as
pertains both to the darkening of these larvae and the complete
expansion of the corial melanophores when placed in the pars

18 The pars intermedia substance was secured by carefully clipping off pieces of
this glandular substance from the subjacent neural lobe, as described by Herring
(14) and Atwell (18). After drying on a warm plate the powdered substance was
triturated with the desired amount of tap-water.

THE PARS BUCCALIS OF THE HYPOPHYSIS 35

intermedia solution. Such a darkening clearly occurs, but an
attendant color change was unnoted by Atwell. These larvae
not only become darker, but they almost completely lose their
metallic iridescent appearance so characteristic of the picture
of albinism. As has been previously stated, this metallic effect
is due to the broad expansion of the xantholeucophores, an
effect which is displayed in its full beauty only because of the
deficiency in the epidermal melanin which characterizes albinos.
As might be surmised, then, these ‘interference’ cells have suf-
fered a great contraction, causing an almost entire loss of the
metallic-like albinous picture. Still another pigment change is
wrought by the pars intermedia solution. The epidermal melan-
ophores undergo expansion, ultimately assuming the expanded
condition typical of the normal animal under ‘indifferent’ en-
vironmental conditions. It is thus seen that the pars intermedia
emulsion can be described as bringing about a normal physio-
logical state in this atypical pigment system of the albino and,
as will be later pointed out, aids us in determining the endocrine
locus responsible for this pigmentary disturbance.2°

When large albinous or normal specimens are placed in an
adrenalin”! solution, serious respiratory and cardiovascular symp-
toms develop. The respiration becomes labored and slowed
and the heart beat decreases in rate. Although more active for
a few minutes, they soon become sluggish and rest in any posi-
tion. Gradually the gills become flushed and it is noted that

1 Two strengths of pars intermedia solutions have been used. One made by
triturating 10 mg. of the dried substance in 100 cc. of water, the other by triturating
10 mg. in 300 cc. of water. Both solutions produced identical effects, but at a
different rate. The larvae would later succumb when left in the stronger solution
for a period of one and a half hours or longer.

2” The immersion of larvae in Pituitrin (Parke, Davis & Co.) has produced
neither constant nor pronounced changes in the pigmentary system even when used
in strengths sufficient to cause the serious distress or death of the animals. An
emulsion of the posterior lobe (less the pars intermedia), even when used in strengths
greatly in excess of that employed with the pars intermedia, appears not to evoke
this reaction. Solutions of hypophysial colloid cause no pigmentary response.

2 Adrenalin (Parke, Davis & Co.) has been employed in strengths of 1 to 20,000
to 1 to 80,000. The stronger solutions (1 to 20,000 and 1 to 40,000 in amounts of
10 to 40 cc. per animal) have been of more value, and attention is called only to
the responses evoked by these strengths.

36 PIGMENTARY GROWTH AFTER ABLATION OF

the entire animal appears somewhat translucent. While evoked
to an extent by the lower dilutions, these effects are much more
clearly expressed with the stronger dosages. If the animals
be examined with the binocular at five-minute intervals, the
progressive alterations in the pigment cells can be followed.
Especially uniform and particularly well pronounced is the con-
traction of the deep chromatophores. This contraction is the
most important factor in the translucent appearance which
follows the administration of adrenalin.

In adrenalin treatment the superficial melanophores are also
affected, those of the albino more uniformly than those of the
normal. In the albino these cells gradually expand and after
an average period of forty to forty-five minutes a very changed
state is evident. The picture presented is the reverse of that
which obtains normally. Instead of these cells for the most
part being in a contracted condition, they now show a state of
expansion. Although only a part of this cell type is completely
expanded, the percentage of completely expanded cells has
definitely increased and all exhibit a more expanded state than
before treatment. In the large normal animal the response of
these cells when a considerable amount of fluid is used (40 cc.
per specimen) is one of expansion. If only partly expanded,
the processes soon present their maximum size; if already com-
pletely expanded, this condition persists. With a small amount
of fluid (10 cc. per animal) these effects are not so pronounced
and, curiously, are not infrequently reversed.

The third group of pigment cells, the xantholeucophores, also
respond to adrenalin. In the albino the surface layer of these
cells is the first to withdraw its processes, a response which
may or may not extend to their more deeply placed brothers.
Indeed, not all of these cells of the superficial stratum are af-
fected, for here and there are interpolated areas of uncontracted
cells, and a maximum contraction of all these cells has not been
secured in the large albino, save with a lethal dose. In the
normal, these cells react in a most varied manner, their reac-
tion, as with the epidermal melanophores, apparently depending
upon the amount of fluid used. With the larger amount there

THE PARS BUCCALIS OF THE HYPOPHYSIS BY

is usually a contraction, although sometimes preceded by a
preliminary expansion. With the smaller amounts these cells
usually expand, there then being presented in the more favorable
cases xantholeucophores of approximately the same size, both
in the albino and in the normal.

The deep melanophores of both albinous and normal larvae
contracted by a solution of pineal glands (two beef glands in
150 ce. water), the same transparency being exhibited as after
adrenalin.” This contraction of the deep melanophores, which,
as pointed out, can be produced by darkness as well, somewhat
intensifies the albinous appearance of true albinos, but its ex-
treme effect in no case renders normal larvae albinous in appear-
ance. These facts are of service in the evaluation of the relative
significance of the various groups of pigment cells in producing
the picture of albinism.

The anatomical and physiological findings which have been
detailed may now be reexamined in order that we may be able
to evaluate the relative importance which the various com-
ponents of the pigmentary system have in creating the picture
of albinism. Any explanation of structural or functional causes
for the albinous appearance must keep clearly in mind the ar-
rangement of these pigment cell types. It will be recalled that
the deepest group of pigment cells—the corial melanophores—
are overlaid by double strata (in most regions of the body) of
xantholeucophores, which in turn lie beneath the epidermal
pigment formed by the double component of free melanin and
epidermal melanophores. With this arrangement in strata it
is obvious, then, that a decrease in the color depth of an over-
lying stratum will exaggerate the effect of the subjacent one,
or conversely an increase in the color depth of a stratum will
proportionately decrease the color effect of those lying beneath.
The decrease in pigment which obtains in the superficial pig-
ment layer (epidermal) of the albino then exaggerates the chro-
matic effect of the abnormally expanded subjacent strata of the

22 With neither of these agents was the extreme transparency gained which Mc-
Cord and Allen describe. Indeed, it is difficult to conceive how such an extreme
effect could be produced with the very considerable amount of melanin which the
epidermis of these specimens contains.

38 PIGMENTARY GROWTH AFTER ABLATION OF

xantholeucophores, whose abnormal expansion in turn masks
or minimizes the effect of the underlying layer, the deep melano-
phores. The mere arrangement which obtains, together with
the physiological and structural modifications met in the albino,
tends to exaggerate the effect of the ‘interference’ cell layers,
and to minimize the réle of the melanin-containing cells (the
epidermal melanin being structurally deficient, the deep melano-
phores being masked by the overlying, broadly expanded layers
of xantholeucophores), hence the color of the albino is char-
acterized by its iridescent metallic nature. This effect is different
from the transparency displayed by dark adapted normal larvae
or those under pineal or adrenalin treatment, procedures which
contract the deep melanophores and, since the xantholeucophores
are unexpanded, enable this effect to be seen in a general lighten-
ing of tone (translucency) of the larvae.

It is possible to still further test the validity of this reasoning
by examining the results of our experiments in which larvae
were submitted to various stimuli (environmental and pharma-
cological) which brought about changes in the components of
one or more of these pigment cell layers. A pronounced darken-
ing of the albino was caused by immersion in pars intermedia
solution. This change, however, does not permit us to analyze
the réle played by the various pigment strata, since all are
affected (it will be recalled that the deep and superficial melano-
phores expand and the xantholeucophores contract, the epi-
dermal free pigment of necessity not being changed). Yet. by
applying a test by which the pigment cells, save for the xantho-
leucophores, undergo the same change as with pars intermedia
emulsion, we can determine whether the expansion of the deep
and superficial melanophores has materially contributed to this
darkening and loss of the metallic tone.23 Such a test is afforded
by placing albinous larvae in intense light when it is seen that
they do not materially darken in color, although the deep melano-
phores may have been previously contracted. The translucency
resulting from a maximal contraction of these cells has pre-

*8 The epidermal melanophores are so scanty in number and pigment content
that their expansion a priori could have little effect.

THE PARS BUCCALIS OF THE HYPOPHYSIS 39

viously been noted. It thus appears evident that the réle of
the deep melanophores in the formation of this picture of al-
binism is of no great importance. In fact, they are so completely
masked in the body region in the albino by the overlying, broadly
expanded xantholeucophores that from the anatomical arrange-
ment alone it would be apparent that in R. boylei they could
play no significant réle in the formation of this picture, and most
certainly not the primary réle as maintained by Atwell in R.
sylvatica.

It is also possible to test the relative significance of the dimin-
ished pigment content of the epidermis in the formation of this
albinous picture. Attention has been called to the partial
replacement of the pigment deficiency which takes place with
a posterior lobe diet, and it was seen that these larvae became
distinctly darker in color than the albinos not supplied with
this gland. Or again, in the skin exchanges, the normal graft,
whose xantholeucophores become broadly expanded, neverthe-
less is decidedly darker than the albinous host. This color
depth can be referred in both cases in part to the color directly
contributed by this epidermal melanin and in part to the masking
of the underlying xantholeucophores by this pigment. It thus
seems clear that the anatomical factors chiefly involved in the
picture of albinism are, first, the broad expansion of the xantho-
leucophores; second, the diminution in the epidermal melanin
which permits full display of the refractive xantholeucophores.

The greatest interest attaches to the determination of the
endocrine locus responsible for the altered structural and physio-
logical state of the pigment cells. Attention was called to the
fact that the feeding of neither anterior lobe, adrenal cortex,
adrenal medulla, nor liver deepened the color of the albino.
On the other hand, a diet of posterior lobe (and associated pars
intermedia) materially darkened the albino—an effect immedi-
ately referable to a partial replacement of the epidermal melanin.
Also the immersion of the larvae in a solution of pars intermedia
alone, of all the endocrine extracts employed, produced a normal
physiological condition of the chromatophore system. It would

40 PIGMENTARY GROWTH AFTER ABLATION OF

thus appear from these responses that the extracts or diets of
the intermediate and posterior lobes of the pituitary were inti-
mately concerned in this pigmentary fault. Corroborative evi-
dence is supplied by a study of the structural malformations
occurring in the endocrine glands of the albinous and the par-
tially hypophysectomized larvae. For, as will be detailed in a
subsequent section (4), the neural hypophysis, thyroid, epi-
thelial bodies, adrenal cortex, and adrenal medulla suffer definite
and often profound structural and size alterations in the albino.
In the partially hypophysectomized tadpole, however, whose
pigment system may suffer a profound disturbance (indeed, the
picture of albinism approaching that of the true albino), none
of these internal secretory organs save the posterior lobe and
the associated pars intermedia exhibit significant structural
‘defects.24 The evidence thus points to a deficiency in the secre-
tion formed by either the posterior or intermediate lobes of the
pituitary or by the ‘interaction’ of these two intimately asso-
ciated components of the pituitary as being responsible for the
pigmentary upset in the larvae.?*

24 Further evidence freeing the thyroid from responsibility for this pigmentary
disturbance comes from the thyroidectomized specimens whose pigmentary system
is not at fault.

26 Indeed, the pigmentary system in the frog, at least, appears to be very sensitive
to hypophysial disturbance. This is shown in a striking way by one specimen of
a considerable number (seventy) of tadpoles which suffered the extirpation of their
pineal glands in an early larval stage. In this operation the aqueduct of Sylvius
of one specimen was inadvertently injured, an injury leading to its complete occlu-
sion. This apparently induced an internal hydrocephalus of the III and lateral ven-
tricles with the consequent inevitable pressure upon the pituitary, sections revealing .
a flattened gland slightly reduced in size. The specimen exhibited unmistakable
albinism,’a condition unquestionably referable to the compression of the pituitary
gland, since it is certain that this pigmentary disturbance was not due to the loss
of the pineal body, for, as has been shown by Laurens (13), the loss of this gland

produces no fault in the pigmentary system, ample confirmation of which has been
made in this laboratory.

THE PARS BUCCALIS OF THE HYPOPHYSIS 41
3. GROWTH DISTURBANCES

Little doubt can be entertained but that the pituitary gland
plays a significant rdle in the regulation of growth. Observa-
tions on clinical material which have been elucidated and ex-
tended by experimental operative work, more particularly by
that of Paulesco and Cushing, have shown rather clearly that a
hypersecretion of this gland results in an overgrowth of the
skeletal system (giantism and acromegaly), the opposite picture
of a pituitary deficiency being seen in dystrophea adiposo-
genitalis.

Clear as such findings appear to be, it seems the more enig-
matical that the administration of this gland has failed to elicit
a definite growth accelerative response, indeed the response
most uniformly evoked being one of retardation. The evidence
so far presented is in many cases at least of such an equivocal
and uncertain nature as to come well within the limits of experi-
mental error and to even be subjected to an opposite interpre-
tation. This can be said not only of the feeding experiments
of different investigators, but of single investigators as well,
and only to the latter do we need to address our attention for
a moment. Aldrich (’12), from the administration of anterior
lobe to pups, concluded that growth was not stimulated nor
retarded, but later concluded from work on white rats that growth
retardation occurred (Aldrich, ’12). Robertson, in the most
elaborate work which has yet appeared upon this subject and
who has not only used large numbers of animals of the same
species (white mice), but who has also applied the valuable
adjuvant of refined statistical methods to his data, at first
concluded (Robertson, ’16), ‘‘The administration of 0.125 gms.
per day per animal of fresh anterior lobe pituitary tissue to
mice, beginning at 4 weeks after birth (conclusion of the
second growth cycle), leads to retardation of growth during the
earlier portion of the third growth cycle, between the 6th and
20th weeks. In the latter part of the third growth cycle, how-
ever, from the 20th to the 60th weeks after birth, the growth
of the pituitary-fed animals is markedly accelerated, so that they

42 PIGMENTARY GROWTH AFTER ABLATION OF

not only catch up to the normals, but actually, at about one
year of age, come to surpass the normals in weight.’’ Later
he says (Robertson, 719): ‘‘The computation of the deviations
in terms of probable error units shows that the effect of the
dosage of pituitary tissue administered upon the growth of
the male animals was of uncertain significance, since the observed
deviations were only from one to two times the probable error.
That the deviations from the normal nevertheless were real
and due to the administration of the pituitary tissue is evidenced
by the much greater effect of the same character upon the female,
consisting of a retardation during the earlier stages of growth.
In both males and females the deviations from the normal after
the 30th week are of indeterminate significance; that is, the
growth curves of the normal and of the pituitary-fed animals are,
so far as our estimates reveal, identical after seven months of
age. Hence the preliminary retardation of growth has clearly,
prior to the 30th week, been succeeded by acceleration.” That
a transient growth retardation in the young animal results from
pituitary administration seems evident not only from the work
of Robertson (’16, 719), but from that of Cerletti (07), Sandri
(09), Aldrich (12), Wulzen (14). But that any subsequent
acceleration which may be exhibited is due to anterior lobe
administration is not clear. Indeed, it seems not improbable
that such an acceleration is compensatory, such as Osborne
and Mendel have shown to occur with an adequate diet after
an animal has been stunted by undernutrition. This seems the
more probable in view of Robertson’s feeding experiments with
Tethelin in which he was forced to abandon the view which
he had previously strongly urged—that his alcoholic anterior
lobe extract (Tethelin) supplied a growth-accelerating principle.
These animals, indeed, exhibited a more pronounced secondary
acceleration when the Tethelin diet was withdrawn than when
it was continued.

The principle has been formulated by Halsted that a ‘physio-
logical deficit’ is essential for successful organ transplantation.
From analogy it seems not improbable that if a physiological
deficit could be created in experimental animals, they would

THE PARS BUCOALIS OF THE HYPOPHYSIS 43

be able to ‘utilize’ the growth-maintaining (‘accelerating’) prin-
ciple of the administered anterior-lobe substance as they could
not do were this deficit not existent. If, then, the animals
suffering from such a deficit exhibited a pronounced retardation
in growth and if in other animals this retardation could be pre-
vented by pituitary administration, we would obtain a com-
bination of conditions giving a pronounced effect from such a
dietary régime. Such indeed is the case in the hypophysec-
tomized (albinous) frog tadpole, as has previously been pointed
out by the author (Smith, 18). The growth response to an-
terior lobe administration in such animals, as compared to the
normal, is greatly magnified, since, 1) the hypophysectomized
frog tadpole grows at a slower rate than the normal (Smith,
Allen) and never attains, or attains only after an extended
period, the size of the normal tadpole (Smith) and, 2) the oper-
ated tadpole supplied with bovine anterior lobe not only ex-
hibits a normal growth rate, but, due to the persistence of the
larval condition and the consequent extension of the larval
growth span, ultimately attains a size notably in excess of the
normal. By this sensitiveness of the albinous frog tadpole a
clear response to the growth-maintaining principle of the pituitary
has been secured.?¢

The ease of securing such operated material, its prolonged
survival after buccal hypophysectomy, and the magnified re-
sponse of the albinous frog tadpole to anterior-lobe adminis-
trations all combine to make this material of singular value in
testing various hypophysial extracts and residues for the pres-
ence or absence of the growth-maintaining (‘accelerating’) prin-
ciple. Such material alone affords an ideal way to analyze
physiologically the various hypophysial substances which have
been .administered in order to determine the réle of this gland

% It will be quite evident why the term growth-‘maintaining’ substance is used
in comparing the growth rate of the albinous larvae with the growth rate of the
normal. The growth rate of the albino is not accelerated by the administration
of anterior lobe as compared to the normal; their growth rate is maintained. Such
a diet, however, clearly accelerates the growth of the albinous tadpole as compared
to their albinous fellows on liver diet.

44, PIGMENTARY GROWTH AFTER ABLATION OF

in growth.?? It is obvious that a determination of the presence
or absence of a growth-‘accelerating’ principle in an extract
or fraction of this gland can hardly be made with the normal
animal when the administration of the fresh gland does not call
forth such a response.

The growth rate of the albino; the effect of the administration of
various hypophysial substances upon this growth rate

In order to discuss the deviations from normality of the growth
~rate of the hypophysectomized tadpole and the effect of the
administration of fresh bovine anterior lobe and its extractives
and residues upon these deviations, it will first be necessary to
examine the growth rate of the normal unoperated tadpole.
The growth curve of a normal tadpole with a liver-lettuce diet
consists of three phases, the first two indistinctly separable,
the third more pronounced. An early transient and not clearly
marked period of slow growth, which perhaps should receive
even less emphasis than has been accorded it (Smith, ’18), shades
insensibly into a protracted second phase of rapid growth during
which the tadpole attains nearly its maximum size, and which
is in turn terminated rather abruptly by the advent of meta-
morphosis (fig. 1). The administration of bovine anterior lobe
neither alters the nature of this curve nor produces quantitative
results of unquestionable significance, although there appears
to result in most cases from this diet a small increment in size
and a somewhat earlier completion of metamorphosis.

The growth curve of albinous frog larvae with a liver-lettuce
diet differs in character and in magnitude from that of the nor-
mals (fig. 1). I have described these changes previously (Smith,
18), and later work has amply confirmed them. A retardation
in growth following the appearance of albinism is unquestioned
and becomes progressively greater until approximately a 30- to

*" Caselli (’00), whole-gland glycerin extracts; Cerletti (01), a centrifugalized
aqueous-glycerin emulsion; Aldrich (’12), fresh desiccated non-defatted anterior-
lobe substance; Aldrich (’12),. fresh defatted anterior-lobe substance; Robertson

(16), fresh anterior lobe and his alcoholic extract, Tethelin, of the desiccated an-
terior lobe.

THE PARS BUCCALIS OF THE HYPOPHYSIS 45

607 FROG
TADPOLES.

I@ntmuous diet of thresh
lonteriar lobe\ inst tuted

12
20 7
307

on 8 o eB Rongum cnhae tad © a
> wo = & & si Fa
4 z= a a w 0 Zz
= =) 2 « a
5
2

Fig. 1 Growth curves of normal frog larvae supplied with a liver diet, hypophy-
sectomized frog larvae supplied with liver and with fresh anterior-lobe substance.
The ordinates represent the total length of the larvae expressed in millimeters.

46 PIGMENTARY GROWTH AFTER ABLATION OF

32-mm. stage (midlarval period) is reached. At this stage the
retardation becomes extremely pronounced and an abrupt change
in the direction of the growth curve ensues. So definite and
so characteristic is this point that it appears to indicate a sig-
nificant period in the development of these albinous larvae and
hereafter will be designated’ as the ‘critical point.’ Both in
time and stage of development this point has been coincident
in the curves plotted from data secured in 1918 and 1919. From
this ‘critical point’ the divergence between the curves of the
normal and albinous larvae increases for a time, the curves
later approaching each other again due to the onset of meta-
morphosis in the former and the continued growth of the latter.

It is now possible to inquire whether the administration of
a ‘substitution’ or ‘replacement’ diet of fresh bovine anterior
lobe will effect a normal rate of growth in these animals suffering
from hypophysial deprivation. This can be answered for the
frog tadpole in the affirmative. The albinous frog tadpoles sup-
plied throughout their growing period with a continuous diet of
fresh anterior-lobe substance exhibit no significant growth devia-
tion from the liver-fed normals (fig. 1, table 2). Since meta-
morphosis does not supervene, however, their growth extends
beyond the normal larval period, and they consequently attain
a size notably in excess of their unoperated normal fellows.
The alimentary régime of fresh bovine anterior lobe has then
clearly supplied the morphogenic principle lost by buccal hypo-
physectomy.

It is conceivable that pituitary substance greatly in excess
of the amount actually requisite to replace the growth principle

28 It will be noted from the tables that after September 7th a continuous diet of
fresh bovine anterior lobe was supplied the albinos whose curve of growth is here
plotted. This apparently caused an abrupt rise in their growth curve. The growth
curves of a group of albinos supplied throughout their life-span with a liver diet are
shown on page 18 (Smith, ’18).

* The final and rather abrupt termination in growth exhibited by the albinous
specimens might conceivably be due to one of at least two factors: 1) a self-limiting
growth factor inherent in every organism; or, 2) a seasonal growth rhythm. That
this is not attributable to the latter is shown by a group of albinous frog larvae
which survived for seventeen months. No resumption of growth occurred in the

second season; rather, they became progressively more lethargic, the anorexia in-
creased, and death finally supervened.

47

THE PARS BUCCALIS OF THE HYPOPHYSIS

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PIGMENTARY GROWTH AFTER ABLATION OF

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THE PARS BUCCALIS OF THE HYPOPHYSIS

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50 PIGMENTARY GROWTH AFTER ABLATION OF

lost by hypophysectomy has been supplied these albinous larvae
exhibiting a normal growth rate, since it will be noted that this
anterior-lobe substance was fed daily and formed their entire
food supply aside from a small component of lettuce. This
seems the more probable if the conception usually held in regard
to ‘hormonal’ substances be correct, namely, that minute
amounts are effective. But that this substance must be sup-
plied continuously is clearly shown by three groups of albinos:
1) a group receiving anterior-lobe substance once each week,
liver being fed on the other days; 2) a group receiving anterior
lobe until an average length of 36 mm. (on July 18th) had been
attained, a liver diet then being instituted; 3) a group (the
converse of group 2) reared on a liver diet until they had attained
an average length of 33 mm. (on September 7th), the liver diet
then being replaced by anterior lobe.

The growth curve of the first group, those receiving anterior
lobe once a week, although considerably modified, resembles
that of the liver- rather than that of the hypophysis-fed group
(fig. 2). Though the incidence of the early retardation is slightly
deferred, nevertheless the ‘critical point’ appears in the usual
position, though in a less pronounced manner. After a brief
period of depression, the curve rises in a regular manner until
the level of the normal is attained. Their curve thus resembles
that of the liver-fed albinos, and the ultimate size attained by
these specimens is intermediate between that of the anterior-
lobe- and that of liver-fed specimens.

The second group, those receiving anterior lobe until July 18th,
grew at a normal rate up to and for approximately two and
one-half weeks past the time when the liver régime was insti-
tuted (fig. 2). A definite growth retardation then appeared,
the specimens eventually attaining the size of their unoperated
companions, but failing by some 10 mm. to reach the size of
their operated mates supplied throughout their growing period
with glandular hypophysis.

The specimens of the third group, those supplied with liver
until they had attained a size of 33 mm., had suffered the usual
early growth retardation and the pronounced midlarval slowing

THE PARS BUCCALIS OF THE HYPOPHYSIS 51

607 FROG
TADPOLES .

7 14 Ler diet a
al Sastitubed ff

284
249
20 7
307

4
2t 4
Oct 6 4

"4
'7

249
34

JUNE 3 4

(24

20 4

307

JULY 6 5
3

207

277

AUG,3 4

SEPT.7 4

Nov.2 7

Fig. 2. Growth curves of hypophysectomized frog larvae supplied with a con-
tinuous diet of fresh anterior-lobe substance, anterior lobe once a week and anterior
lobe from May 6th to July 18th, then liver. The ordinates represent the total
length of the larvae expressed in millimeters.

52 PIGMENTARY GROWTH AFTER ABLATION OF

invariably exhibited by albinos supplied with this diet. Follow-
ing the substitution of a hypophysial for a liver diet, a normal
growth rate is exhibited for a time, the albinos, however, not
attaining the large size induced by continuous anterior-lobe
feeding (fig. 1).

It seems clear from these three groups that neither an inter-
rupted diet of anterior-lobe substance nor a full diet of this
substance during merely the early or the late portions of the
growing period will bring about a normal growth of the albinous
larvae. It is indeed remarkable that albinous larvae, growing
at a normal rate in response to anterior-lobe administration,
will exhibit a growth retardation so quickly on the withdrawal
of this ‘substitution’ diet.

It may then be regarded as demonstrated that the retarda-
tion in growth, resulting from the early removal of the epithelial
hypophysis, can be ameliorated by a continuous diet of bovine
anterior lobe, the growth curves of the albinous larvae thus
treated showing no deviation from normality save by its con-
tinuance beyond the normal growing period. It will likewise be
granted by the reader that such material is of peculiar value for de-
termining the presence of the growth-maintaining principle in any
hypophysial extractive or its residue. The experimental utiliza-
tion of this material should show us whether the extraction of the
fresh anterior lobe with boiling alcohol or boiling water removes
or destroys this growth-maintaining principle—a test which can
be provided with a double check, since both the extract and its
residue can be fed to similar groups of albinos. To the group of
four substances (aqueous extract and its residue, alcoholic ex-
tract and its residue) which were submitted to this test was
added the hypophysial colloid occurring in the intraglandular
(vestigial cleft) of this gland.#+

30In the preparation of the aqueous extract and its residue, the ground fresh
anterior-lobe substance was extracted in a modified Bailey-Walker apparatus (Rob-
ertson) over boiling aq. dest. for forty-eight hours, five bovine anterior lobes being
placed in each alundum thimble. After this prolonged extraction the substance
was dried, powdered, and placed in vials tightly corked. Each group of specimens

(20 to 24) received the dried residue from one and a half glands at a feeding. The
clear, yellowish extract on evaporation yielded a dark, readily soluble amorphous

THE PARS BUCCALIS OF THE HYPOPHYSIS 53

Obviously, since the aqueous and alcoholic extracts and the
colloid were of slight or no nutritive value, additional food
substance had to be furnished, but such food substances were
only supplied after obtaining a maximum intake of the special
glandular substance. After turning off the flowing water, the
special substance was fed, one half-hour later the nutritional
substances, liver and lettuce, being supplied. Twenty-four hours
later, after removing all food particles, the process was repeated.

In the accompanying table (table 3) is shown the number
of frog larvae at the commencement of the special feeding and at
three subsequent periods. Although the number is relatively
small, yet they were average-sized, carefully selected, healthy
tadpoles free from injury or malformation. Work with a larger
number was precluded, due to the difficulty of securing a suf-
ficient amount of the special substances, 90 to 100 bovine hypo-
physes being used daily with even this number of specimens.

substance giving a lipoid-like odor on burning. The extract from five glands was
supplied each group of tadpoles (20 to 24) at a feeding. The feeding was done
with sufficient rapidity to insure against an extensive solution of the solid substance,
and from the rapidly initiated movements of the tadpoles it was certain that they
were feeding directly upon this substance.

The alcoholic extract (Tethelin) was supplied by H. K. Mulford Company. In
the feeding of this substance even greater care was exercised than with the aqueous
extract, to guard against solution. After a period of a few seconds subsequent to
the breaking of the vacuum-sealed tube, the tadpoles would be feeding upon the
substance. Fifty milligrams (the extract from five glands) was given each group
at feeding.

In the preparation of the alcoholic residue the method elaborated by Robertson
for the extraction of Tethelin was slightly modified. Dehydration was carried out
with 95 per cent alcohol instead of anhydrous sodium and calcium sulphates, since
the addition of the salts would render the residue useless. Following dehydration,
the tissue was thoroughly dried over an electric stove, then extracted in a Bailey-
Walker apparatus with boiling absolute alcohol for forty-eight hours. The residue,
freed from alcohol, and preserved in tightly corked vials, was supplied in amounts
equal to one and a half of the fresh glands at a feeding.

The colloid was secured from the vestigial cleft, between the glandular and inter-
mediate lobes. The amount obtained from the forty-five glands and administered
to the twelve to fifteen tadpoles at each feeding varied considerably, but averaged
0.5 gram. Occasional very large pieces (0.675 gram) were found, many other
glands revealing none. Usually it occurred as a firm, amorphous mass, molded
by the shape of the cleft, now and then, however, gels of differing viscosity or even
an aqueous-like substance was obtained. Before feeding, the amorphous masses
were crushed and mixed with the more fluid portions.

OF

TH AFTER ABLATION

PIGMENTARY GROW’

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THE PARS BUCCALIS OF THE HYPOPHYSIS 55

Further, it was believed that if this number of animals should
show an unequivocal response to hypophysial administration,
it would furnish convincing evidence of either the presence or
the absence of the growth-maintaining principle in these special
substances—a belief which we feel has been amply sustained.

The growth curves of the albinous frog tadpoles supplied
with either alcoholic or aqueous residues show the same main-
tenance of growth as is exhibited by their fellows fed with the
fresh anterior lobe (figs. 3, 4). No ‘critical point’ appears;
the three curves are coincident.

This is not the case with frog albinos receiving aqueous ex-
tract, Tethelin, or colloid (figs. 3, 4, 5). Their curves are quite
similar to that of the liver-fed albinous larvae. The same early
retardation is noted, the ‘critical point’ is well marked, but
appears at a somewhat earlier period (twelve days) and at a
somewhat smaller average stage (6 mm.) than their liver-fed
companions. Following the critical point, some divergence ap-
pears in the three curves. The specimens supplied with aqueous
extract exhibit a slight secondary acceleration in growth, but
they do not attain quite the average length of their liver-fed
mates. The ‘Tethelin’ group does not exhibit any secondary
acceleration subsequent to the ‘critical point,’ while the colloid
group exhibits an even greater retardation than its fellows sup-
plied with Tethelin.

In order to further test the growth effects of hypophysectomy
and its response to an anterior-lobe diet, a series of experiments
similar in every way to those just reported upon were carried
out with toad larvae. These larvae respond in general in the
same way as the similarly treated frog tadpoles. The early
retardation, the usual midlarval slowing (‘critical point’), fol-
lowed by slow growth, appears as in the frog tadpole (fig. 6).
In this, my observations are somewhat at variance with those
of Allen, who reports no retardation in the velocity of growth
in hypophysectomized toads. Fuller reports may explain this
essential difference in our results. I wish to emphasize, how-
ever, that the early retardation, the ‘critical point,’ and the
later period of slow growth are not so distinctly shown in the

56 PIGMENTARY GROWTH AFTER ABLATION OF

607 FROG
584 TADPOLES aa

4
234
JUNE 3 J
2
20 4
307
JULY G
13
20 4
27 7
AUG 3
"oy
\7 4
24 4
31
SEPT.7 7
1+ 4
a4
28
ocr.6 5
2 4
204
NOV. 2 4
30

Fig. 3 Growth curves of hypophysectomized frog larvae supplied with fresh
anterior-lobe substance, with aqueous extract, and with aqueous residue of the
anterior lobe. The ordinates represent the total length of the larvae expressed in
millimeters.

THE PARS BUCCALIS OF THE HYPOPHYSIS 57

FROG

TADPOLES
cai aes,

.
A 5. nos Alcoholic extract
/ iT
o q sd

3

234

JUNES 9
12
20 4
30
JULY 6 4
3
20 4
27 4
AUG. 3 5
"4
(7
244
3
sePt.7 4
4 4
214
28 4
oct.6 4
le 4
20 4
Nov.2 4
so

Fig. 4 Growth curves of hypophysectomized frog larvae supplied with fresh
anterior-lobe substance, with alcoholic extract and with alcoholic residue of the

anterior lobe.
millimeters.

The ordinates represent the total length of the larvae expressed in

58 PIGMENTARY GROWTH AFTER ABLATION OF

604 FROG
584 TADPOLES. t—>~ =

of artract.
albinos 1%y ee

9.070 Od

134
234
12 4
204
304

JULY 6 4
3
204
274
WF
'T
244
34
44
a4
24
4
204
304

MAY 6
JUNE 3 4
AUG.3 4
SEPT.7 4
oct. 64
NOV.2 4

Fig. 5 Growth curves of thyroidectomized frog larvae supplied with liver diet,
hypophysectomized frog larvae supplied with fresh anterior-lobe substance, and
with hypophysial colloid. The ordinates represent the total length of the Iarvae
expressed in millimeters.

THE PARS BUCCALIS OF THE HYPOPHYSIS 59

607 TOAD

584 TADPOLES. go
q

Nov24

Fig. 6 Growth curves of normal toad larvae supplied with fresh anterior-lobe

substance, hypophysectomized toad larvae supplied with fresh anterior-lobe sub-

stance and with liver. The ordinates represent the total length of the larvae ex-

pressed in millimeters.

60 PIGMENTARY GROWTH AFTER ABLATION OF

toad as in the albinous frog larvae. This is also true of their
response to a ‘substitution’ diet of anterior-lobe substance.
The growth rate of those toad albinos supplied with an hypo-
physial diet does not quite equal that of their normal brothers.*
Neither has the administration of hypophysial extracts, residues,
and colloid given as clear results as in the frog tadpole (figs. 7,
8, 9). However, an unmistakable growth retardation is shown
by the albinous toad larvae supplied with the extracts and
colloid, and the administration of the residues has caused a
distinct acceleration in growth rate as compared to their liver-
fed albinous mates. Table 4 gives the measurements upon which
the growth curves of the toad tadpole are based, and table 5,
the number of specimens used in the study.

From a survey of these groups receiving the various hypo-
physial substances and liver it is evident that they are separable,
whether frog or toad larvae be used, into two distinct cate-
gories, both by the size attained and by the nature of their
growth curves. On the one hand is the group formed by those
larvae supplied with either the alcoholic or aqueous residues;
their growth curves are identical with that of the fresh anterior-
lobe-fed group; on the other hand is the group formed by the
larvae receiving Tethelin, aqueous extract and colloid; these
exhibit some variation in size, but their curves are similar in
nature to that of the liver-fed albinos.

To what factor or factors is due, 1) the normal growth rate
of the albinous larvae supplied with fresh anterior lobe and
the hypophysial residues, and, 2) the retardation of specimens
fed with liver, hypophysial extractives, and colloid? The nor-
mality of the growth rate exhibited by the specimens of the first
division might conceivably be due to the superior nutritive
value of the anterior lobe rather than to a morphogenic agent.
If this was the responsible factor, however, we should expect as
great a deviation in both the magnitude and the nature of the
growth curves of normal tadpoles fed in this way. No such

31 It is to be noted, however, that the normal specimens received a diet of fresh

anterior lobe, not of liver. A slight acceleration in the normal produced by this
diet may account for the divergence in the two curves.

THE PARS BUCCALIS OF THE HYPOPHYSIS 61

605 TOAD

584 TADPOLES. Pall Ns
56

Fig. 7 Growth curves of hypophysectomized toad larvae supplied with fresh
anterior-lobe substance, with alcoholic extract, and with alcoholic residue of the
anterior lobe. The ordinates represent the total length of the larvae expressed in
millimeters. ,

62 PIGMENTARY GROWTH AFTER ABLATION OF

6074 TOAD
584 TADPOLES. ae
56
as
547
524

——-

71-7
=
S 8s

J04

+
Le}
SS

g

Nov.2 4

Fig. 8 Growth curves of hypophysectomized toad larvae supplied with fresh
anterior-lobe substance, with aqueous extract, and aqueous residue of the anterior
lobe. The ordinates represent the total length of the larvae expressed in milli-
meters.

BUCCALIS OF THE HYPOPHYSIS 63

THE PARS
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expressed in millimeters.

PIGMENTARY GROWTH AFTER ABLATION OF

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MEMOIR NO. 11.

66 PIGMENTARY GROWTH AFTER ABLATION OF

effect has been produced in normal larvae by such alimentary
régimes.

One article of diet, liver—the chief nutritive substance sup-
plied—is common to the specimens of the second division.
That the retardation in the growth rate of these specimens is
not due to a growth-retarding substance in the liver, but to the
absence of the hypophysial growth-maintaining substance, is
clearly indicated by three lines of evidence: 1) we should have

TABLE 5
Table giving the number of normal, thyroidectomized, and albinous toad larvae on which
the accompanying growth-curves are based

THYROID
BUCCAL COMPONENT OF HYPOPHYSIS ABLATED ABLATED| NORMAL
* . Glandu-
Liver Liver
; DIET OF GLANDULAR LOBE OF HYPOPHYSIS ; Jar lobe
dict | diet diet
Fresh Alco- | % Alco-
glandu-| holic |.i holic {Aqueous |Aqueous | Colloid
lar lobe | extract | residue | extract | residue
June 25} 8 30 8 8 8 8 8 14 18
Aug. 13) 7 23 7 8 6 8 8 13 18
Sept. 14] 7 21 7 6 6 8 7 13 0
Oct. 20] 6 20 4 6 or 6 7 8 0

1 Killed accidentally.

to likewise assume that such growth-retarding substances were
in adrenal cortex, adrenal medulla, and posterior lobe, for the
same growth delay exists when albinous larvae are fed with
any of these substances; 2) Mendel and Osborne (’18) have shown
that the proteins of liver are adequate for the needs of nutrition
in mammalian growth; 3) normal frog larvae supplied with a liver
diet exhibit an entirely normal rate of growth.

It seems clear, then, that the normal growth rate of the al-
binous specimens receiving fresh anterior lobe or its residues is
due to an alimentary replacement of the growth-maintaining

THE PARS BUCCALIS OF THE HYPOPHYSIS 67

substance normally furnished by the tadpole’s own gland. It ig
significant of the stability of this substance that its efficacy
or physiological nature has not been seriously impaired by the
severe treatment accorded it in the prolonged extraction with
boiling water or absolute alcohol. It would seem certain that
the retardation in the growth rate of the albinos receiving liver,
hypophysial extracts, and colloid is due to the absence of the
morphogenic principle normally furnished by the anterior lobe
of the pituitary. .

The growth rate of thyroidectomized larvae

It has been noted by previous workers (Allen, Hoskins) that
thyroidectomized larvae attained a size much in excess of the
normal and that the pituitary glands of these specimens were
relatively increased in size. Since the larval condition persists
in the thyroidless larvae and since the hypophysis is hyper-
trophied, it might be expected that these specimens would ex-
hibit a more prolonged, and a more rapid rate of growth than
even the anterior-lobe-fed albinous tadpole. By thyroidectomy,
then, there would be obtained tadpoles which would simulate the
rapid growth exhibited at times by the acromegalous human
subject.

Observations, however, made on two groups of thyroidec-
tomized frog larvae, one group (seventy-two specimens) fed on
anterior lobe, the other (fifty-nine specimens) fed on liver, show
that their growth is identical in rate with that of their normal
brothers or with the albinos supplied with a continuous diet
of the fresh anterior lobe (fig. 5). Their large size is never
attained by a more rapid growth rate, but rather, as in the
anterior-lobe-fed albinos, by a prolongation of the growth period.
No variation in growth rate was caused by feeding the fresh
anterior lobe of the pituitary, a non-effect already reported upon
by Allen (18). The enlarged pituitary which these animals
possess does not appear to have supplied any growth stimulus
in excess of the normal.

Nevertheless, a different condition obtains with the thyroidec-
tomized toad larvae, at least when supplied with a liver diet.

68 PIGMENTARY GROWTH AFTER ABLATION OF

This is well shown by the members of one group (fourteen speci-
mens) whose growth rate was more rapid than that of their
normal fellows supplied with anterior lobe substance (fig. 9).
Moreover, they attained a size notably in excess of that reached
by their hypophysis-fed albinous mates.

Evidently, then, in the case of the frog larvae the enlarged
hypophysis, which Allen and Hoskins have shown to result from
thyroidectomy, has not supplied any growth stimulus in excess
of the normal. To what the acceleration in the toad larvae is
due has as yet been undetermined, but that an increased secre-
tion from the hypertrophied pituitary may be an important
factor, as suggested by Hoskins, seems not improbable.

4. MODIFICATIONS IN THE SIZE AND STRUCTURE OF THE
ENDOCRINE ORGANS

It seems probable that a tissue, whether its distribution be
diffuse or localized, is more labile and ‘adaptive’ in its early
formative stage than in its mature condition. An example of
this is seen in the nervous system, which in its mature, highly
differentiated condition loses its capacity for regeneration and
‘adaptation,’ a capacity which it exhibits to a surprising degree,
however, in its early formative stages (Lewis), more especially
when associated tissues can elicit a developmental response
(Burr). It is likely, then, that if a disturbance be experienced
by the endocrine system in its early and embryonic stages, greater
alterations in its members will result than if this upset had been
suffered later in the life of the individual. As has been pre-
viously suggested, the more extensive structural changes which
are manifested by the internal secretory organs of the hypophysis-
free tadpole as compared to the hypophysectomized mammal are
probably referable to the earlier institution of this disturbance.
Extensive structural modifications, as we will show, are invari-
ably manifested in the thyroid, the neural portion of the hy-
pophysis, and the interrenal and chromaffin components of the
adrenal gland subsequent to the early loss of the epithelial
hypophysis, while lesser though distinct changes are shown
by the epithelial bodies. The behavior of the fat-organ in
hypophysial deficiency is also described in this section.

THE PARS BUCCALIS OF THE HYPOPHYSIS 69

The hypophysial components

The endocrine disturbance leading to the abnormal condition
of the epithelial bodies, the thyroid and adrenal glands, sub-
sequently to be described, has of necessity been mediated to
these distant organs by the blood stream. The neural hy-
pophysis, while subjected as are these other glands to this general
endocrine disturbance, has, in addition, been subjected to the
loss of its intimate anatomical associate, the epithelial hypophy-
sis, a loss which in itself might conceivably greatly modify the
neural lobe entirely apart from the general disturbance intro-
duced into this correlative system of organs by buccal hypo-
physectomy. That the intimate anatomical association exist-
ing between the neural and epithelial components, to which
attention has been called by many observers,*? is fundamental
for the elaboration of the secretion of at least the posterior lobe,
finds support not only in the anatomical evidence afforded by
this relationship, but also in the recent work of Schmidt and
May from the chemical side.** As will be pointed out, cor-
roborative evidence bearing upon the significance of this coales-
cence will accrue from this study. It must be kept in mind
that in this experimental work the buccal constituent of this
gland has been removed prior to the union with its neural asso-
ciate, which consequently remains undisturbed, and that the
infundibular process at this time gives no indication of the
structural differentiation which it is to later undergo, so that
any modifications exhibited by the neural lobe (or the infundibu-
lar process) are not referable to any direct operative injury
inflicted upon this member. As will be subsequently pointed
out more in detail, sections and models of these albinos reveal
a profound reduction in size, as well as an atypical shape and

32 Herring, Trautmann, Stumpf, Kohn, Erdheim and Stumme, Vogel, Stendel,
and others.

33 Schmidt and May, by appropriate chemical treatment of the alcoholic hy-
pophysial extract, Tethelin, have secured a substance whose action simulates the
active principle of the posterior lobe, i.e., causes a contraction of the isolated uterus
and produces a vasoconstriction.

70 PIGMENTARY GROWTH AFTER ABLATION OF

abnormal position of the neural lobe.*4 Indeed, this malfor-
mation is not limited to the neural lobe, but is participated in
also by the floor of the infundibular process. This floor, nor-
mally of considerable thickness, is membranous and folded in
the albinous larvae.+*

It is of considerable importance, in view of the conditions
obtaining in the albino, to have clearly in mind the parts of the
infundibular process together with their structure in the normal
animal, and more especially that of the apical and ventral por-
tions. The walls of this process have three divisions:** 1) a
ventromedial portion in contact with the pituitary gland, the
‘pituitary’ wall or floor; 2) a dorsal portion, the ‘saccular’ wall;
and, 3) the lateral extensions of both these walls which form
the shallow lateral concavities, the ‘lateral processes.’ The
neural lobe in its early stage develops near the apex of the in-
fundibular process, and in its fully formed condition is readily
identified as a distinct element attached by a broad surface to
the dorsal wall of the infundibular process, near its ventral
border (fig. 25). An examination of the three portions of the
infundibular process of the normal animal reveals the fact that
these regions are of two structural types. One type, that ex-
hibited by the lateral processes and saccular wall, is mem-
branous and composed of a squamous ependyma with little or
no ectally placed neuropilem. The other type, that forming
the pituitary wall, is thickened and composed of a high columnar

34 The reduction in the size of the neural lobe in the albino was reported in The
Anatomical Record. In the present paper more comprehensive data have been
given.

35 The hypophysis of an anuran larva, in common with other vertebrates, con-
sists of four components, three arising from oral ectoderm, one from the infundibular
process. The development of those lobes derived from the buccal rudiment (the
pars intermedia, the pars glandularis and its paired cephalic processes, the pars
tuberalis) conforms in general to that recently described by Atwell for R. pipiens.
Wax models, X133, of the pituitaries of nine normal larvae, from 17 mm. in length
to the young adult stage have been made (table 6). In addition to these, the neural
lobes of ten albinos, ranging in size from 16 to 51 mm., and the neural and buccal
lobes of nine partial albinos were modeled. The lobes of the pituitary are readily
identified and their relationship to each other is constant (fig. 25, a, b, c), compare
to Atwell’s figure 13, page 81, but considerable variation in their relative size is

shown.
36 The terminology is essentially that of Tilney (15).

THE PARS BUCCALIS OF THE HYPOPHYSIS 71

ependyma with an ectally placed prominent stratum of neuro-
pilem (fig. 28). In the albino the saccular wall and lateral
processes and pituitary wall all exhibit the membranous type
of structure (fig. 29). The pituitary floor, which is normally
in contact with the epithelial hypophysis and which has been
deprived of such contact by the epithelial hypophysectomy,
thus exhibits the same type of structure as those portions not
normally in contact with this epithelial component of the hy-
pophysis. Directly attributable to the membranous character
of this pituitary floor are invariably seen certain pronounced but
irregular folds which appear in this structure in the albino (fig.
26), but not in the normal animal (fig. 25). Although such
foldings are undoubtedly due to the technical procedure incident
to the preparation of this material, yet the membranous nature
of the infundibular floor of the albino is directly conducive to
them, while the firmer structure of the normal infundibulum
prevents them.

The abnormality in the infundibular process in the albino
is not limited to the pituitary floor, for it is very evident that
the neural lobe in the albino as compared to the normal animal
of corresponding dimensions (table 6) is also diminished in size,
a diminution which varies from 40 per cent to 80 per cent and
which transcends very distinctly the considerable variability
which this lobe exhibits in both types of specimens.*? Asso-
ciated with this diminution in size is a profound alteration in
the shape and position of this lobe. Instead of the symmetrical,
transversely placed, dumb-bell-shaped lobe of the normal (fig.
25), this dwarfed lobe is oval, asymmetrical in shape and posi-
tion, and has no great transverse extent (fig. 26). Normally
attached somewhat dorsally to the apex of the iniundibular
process and thus abutting and being attached to its dorsal sur-
face, in the albino it is constantly found grasping the apex of
this process and extends invariably slightly onto the pituitary

7 Tt does not appear from figures 28 and 29 that the neural lobe of the albino is
smaller than in the normal. It must be kept in mind, however, that due to the
difference in the shape of this lobe in the two types that a median sagittal section

passes through its smallest dimension in the normal animal, and through its max-
imum sagittal diameter in the albino (figs. 25, 26). >

7 PIGMENTARY GROWTH AFTER ABLATION OF

surface (figs. 26, 29). This lobe is thus profoundly modified by
the loss of its associate. It attains normal development neither
as regards size, form, nor position.** In its response to buccal
hypophysectomy the reduction of this gland thus aligns it with
the thyroid and the adrenal cortex reduction. Indeed, in those
animals suffering only a partial loss of the epithelial hypophysis,
the thyroid and adrenal glands are usually not altered in develop-
ment, but the neural hypophysis is invariably atypical.

In an earlier section of the paper we have referred to the
type of tadpole in which the removal of the epithelial hypophysis
was incomplete. Since these animals usually manifest definite
pigmentary alterations, we have designated them ‘partial al-
binos.’*» Especial interest attaches to the examination of the
endocrine system, more particularly its hypophysial components
in this type, since we have here an intermediate condition be-
tween a complete deficiency of the epithelial hypophysis and
a normal structure. A study of this form throws much addi-
tional light upon the interesting relationship obtaining between
the neural and epithelial components of the pituitary as well as
supplying evidence bearing upon the endocrine locus responsible
for the pigmentary deficiency.

‘In the partial albinos no uniformity in shape is displayed by
the vestige of the epithelial hypophysis. It is usually oval in
outline and of a variable thickness (figs. 27, 30). Only occa-
sionally do we find it displaying the normal division into glandu-

% The findings of Allen (Abstracts, Am. Assoc. Anatomists, 1917) are distinctly
at variance with those reported here. This author states, ‘‘The pars nervosa of
the hypophysis forms normally in tadpoles, both Rana and Bufo, from which the
anlage of the anterior lobe has been removed.” As I have previously pointed out,
a median sagittal section of the infundibulum usually shows a neural hypophysis
of normal or even enlarged proportions. By modeling this component, unmis-
takable evidence concerning its diminution in size and atypical form is secured,
however.

39T called attention in 1916 (Proc. Am. Assoc. Anat.) to an albinous tadpole in
which a leg development took place. At that time the minute buccal fragment
displayed by this animal had not been noted. Allen, in 1918, called attention to

the complete metamorphosis of certain toad and frog tadpoles with an imperfect
hypophysis and displaying a definite pigmentary deficiency.

THE PARS BUCCALIS OF THE HYPOPHYSIS 73

lar and intermediate components. The size‘? of this vestigial
gland is always considerably less than that obtaining in the
normal tadpole of corresponding dimensions (table 6). As would
be anticipated, this epithelial gland, subjected as it was to a
varying surgical interference, leaves a vestige which shows
great diversity in its position as well as in shape and size. That
the position which this lobe assumes is of paramount importance
in the influence it may exert upon the other glands and upon
metamorphosis, is evident from a correlation of the structural
changes enjoyed by the partial albino with the position which
the buccal fragment has assumed. In the specimens thus far
studied, three positions have been assumed by this fragment:
1) In the first case, this fragment, not separable into glandular
and intermediate components, does not reach the caudal ex-
tremity of the infundibular process, nor does it touch at any
point the true neural lobe (figs. 27, 30). The members of this
group, which includes the partial albinos (p 1, p 2, p 3, p 4,
p 5), all displayed a pronounced albinism. It is to be noted
that none of these animals complete metamorphosis, though,
as in the other groups, their thyroid glands are not atrophic.
2) In the second case, the fragment, not separable into glandular
and intermediate components, does not reach its usual caudal
position, but nevertheless attains a definite though small con-
tact with the true neural lobe. This class includes a smaller
number of specimens (p 7, p 9, p 10), the members of which
display a variable pigmentary disturbance. They complete
metamorphosis, though tardily, and the thyroid glands exhibit
a pronounced colloidal hypertrophy (colloid goiter). 3) In the
third case, the epithelial fragment, which is separable into inter-
mediate and glandular components, assumes the position typical
of the normal animal. One specimen only fell in this class (p 8);
it did not exhibit a pronounced pigmentary upset; it completed
metamorphosis and had a normal thyroid gland. A study of a

40 The metamorphic changes in all the partial albinos, except p 1, which died
from infection, whose hypophyses were modeled (table 6), had ceased for some time
prior to the fixation of the animal. It is certain that the metamorphosis would
never have been completed nor even materially advanced had the specimens sur-
vived longer.

PIGMENTARY GROWTH AFTER ABLATION OF

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THE PARS BUCCALIS OF THE HYPOPHYSIS 77

greater number of ‘partial’ albinos, it is anticipated, will reveal
an overlapping of these classes, but because of the apparent
association between the progress of metamorphosis, the reaction
of the thyroid gland and the position of this epithelial fragment,
such a tentative classification appears justifiable and will be
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The description of the picture presented by the epithelial
hypophysis of the partial albino would be incomplete unless we
added a word in regard to the structural characteristic of this
vestige. Its structure (in the first and second classes mentioned
above) is characteristic neither of the glandular nor the inter-
mediate components of the pituitary. Certain portions are
composed of acidophilic cells, other portions of cells similar to
those normally found in the pars intermedia. The organization
of those cells, however, is typical of the pars intermedia, since
the component cells are neither arranged in definite cords nor
separated by numerous nor large sinusoids (fig. 30).

The response of the neural lobe to partial hypophysectomy
is of even greater value in analyzing the interrelationship exist-
ing between the neural and epithelial components of the pituitary
than was the reaction of this lobe to the complete loss of the
buccal component, as obtains in the albino. In size it is usually
diminished as compared to the normal animal of corresponding
dimensions and stage of advancement (table 6) and at times the
lobe as such is not even recognizable (specimens p 2, p 4).
When present it is asymmetrical in shape and position and fre-
quently encroaches upon the pituitary floor. Of great interest
are the conditions obtaining immediately about the epithelial
fragment. About this fragment there is formed a thickening
of the neural tissue which we might designate as a ‘novel’ neural
lobe. This partially surrounds and grasps the buccal fragment
(fig. 30), an appearance which in part may be explained by the
indentation which this lobe makes in the brain substance. When
the usual neural lobe is absent or greatly diminished in size
(specimens p 2, p 4), there is evident in those specimens that
have been thus far examined a thickening of the pituitary floor

78 PIGMENTARY GROWTH AFTER ABLATION OF

(fig. 80), a condition which strongly suggests that the neural
lobe had migrated cephalically and was attempting to assume
a juxtaposition with the unusually placed epithelial fragment.
This seems the more probable, since in the specimens which have
suffered a complete loss of the epithelial hypophysis there is
always a readily distinguishable neural lobe. That the epi-
thelial hypophysis apparently exhibits not only this ‘attractive’
force upon the neural lobe, but definitely stimulates the growth
of adjacent neural tissue, is shown both by the thickening’ of
the neural tissue about the epithelial hypophysial vestige” in
the partial albinos and by the negative evidence supplied by the
absence of the usual thickening of the pituitary floor and dimin-
ished size of the neural lobe in the albino which displays no
epithelial hypophysis whatever. It thus seems clear that in
the absence of a nearly hormal epithelial component the neural
hypophysis cannot undergo its normal development nor attain
its typical shape or size.*? That the neural lobe can and does
form even in the absence of its associate, however, is clear, and
it seems equally clear that in those specimens in which a suf-
ficient metamorphic stimulus is supplied, whether by the thyroid
or the hypophysis or jointly, that this lobe can attain a very
considerable size.‘

41 Although this neural lobe simulates in structure the typical lobe, nevertheless,
it seems doubtful whether it is able to assume the functions of that lobe. That
it can assume this function appears improbable both from the failure of such speci-
mens to metamorphose as well as from the serious pigmentary disturbance which
they display.

* To what an extent the general disturbance in this correlative system of organs
plays a part is problematical. It is difficult to determine, save by grafts of the
buccal component with the hypophysectomized larvae; an undertaking which has
not been fruitful in the hands of the author. A similar failure to secure significant
effects has recently been reported by the Hoskinses. Likewise, efforts to secure
an apposition of the buccal component and the lateral or dorsal walls of the in-
fundibular process have so far failed. The author hoped to determine by such
misplacement of the buccal component whether these walls would undergo the
hypertrophy normally exhibited by the pituitary wall.

43 No clear evidence is at hand showing whether the buccal lobe would undergo
a normal development in the absence of its neural associate. Suggestive evidence,
however, that it can develop independently is furnished by the work of Haberfeldt
(09). According to this author, the pharyngeal hypophysis, although far remote
from any neural associate, parallels in development and differentiation the pars
glandularis of the hypophysis. ‘

THE PARS BUCCALIS OF THE HYPOPHYSIS 79

A study of the older albinous and partially hypophysectomized
larvae does not throw light upon the developmental process
which has led to the thinning of the pituitary floor and the
diminution in the size of the neural lobe in these specimens.
From such a study we can conceive that one of two develop-
mental processes lead to this anatomical condition. In the first
place, it is possible that a normal differentiation and develop-
ment of these parts may have taken place, so that at some not
* very early period they were structurally identical in both the
albinous and normal larvae. Later, due to the absence of the
association with the epithelial hypophysis, these normally de-
veloped parts underwent an atrophy which led to the conditions
just described. Or, in the second place, it is possible that, due
to the absence of the ‘stimulatory’ force normally supplied by
the epithelial hypophysis, the neural lobe and pituitary floor
even from their earliest stages did not undergo the normal
increase in size. A developmental study of the albino, the
normal, and the partially hypophysectomized tadpole affords
clear evidence that the second hypothesis is correct. An exam-
ination of the divisions (lateral, saccular, and pituitary) of the
infundibular process in an early stage (5 to 10 mm.) reveals the
fact that these parts are nearly identical in structure and that
no differences exist between a normal larva and a larva which
has suffered the loss of its epithelial hypophysis. This infundi-
bular pocket, slightly thinner at its apex where it is formed of
a cuboidal epithelium, thickens near its attachment to the firmer
walls of the brain where it is formed of columnar epithelium.
As development proceeds, the dorsal and lateral walls of this
pouch become progressively thinner in both the albino and
normal animal, and at a 14- to 16-mm. stage their epithelium
is of the squamous type and remains so through their larval
life span (fig. 10). Thus the development of the dorsal and
lateral walls (save the small portion giving rise to the neural
lobe) is identical in the two types of animals. It is in the devel-
opment of the pituitary wall, however, that the differences
between the albinous and normal larvae make their appearance.
This portion, both in the albino and the normal, undergoes for

PIGMENTARY GROWTH AFTER ABLATION OF

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THE PARS BUCCALIS OF THE HYPOPHYSIS 81

a time the same thinning as do the dorsal and lateral walls.
By a 9.5-mm. stage the pituitary wall has become much thinned,
and consequently the various portions of the infundibular process
are approximately of the same thickness (fig. 10) and nearly
structurally identical. From this point a series of changes take
place leading to a very different condition in the pituitary wall
of the two types. In the albino the membranous wall persists.
In the normal this becomes progressively thicker and finally
attains a thickness many times that obtaining in the 9.5-mm.
stage. It is significant that the epithelial hypophysis, although
previously in contact with a small portion of the infundibular
floor prior to a 10-mm. stage, institutes an intimate juxta-
position with the pituitary wall of the infundibular process at
the time when the latter loses its membranous character and
begins to thicken.

Differences between the neural lobe of the albino and the
normal appear at a later stage than they do in the pituitary
wall. The thickening near the apex of the infundibular process
indicating the first formation of the neural lobe makes its appear-
ance at approximately a 14-mm. stage in both the normal and
albinous tadpole,‘! and is nearly identical in position and size
in both types at a 16- to 17-mm. stage. From this stage the
neural lobe in the normal increases concomitantly with the
growth and lateral extensions of the developing pars intermedia.
In the albino, on the contrary, a slower growth (table 6) and
relatively slight lateral extension take place—a process leading
to the small, malformed neural lobe of the albino.

The small neural lobe and the membranous pituitary wall
of the albino are thus clearly referable to a non-development
rather than to an atrophy of a normally sized structure. The
walls of the early infundibular process have apparently two
inherent possibilities after having undergone the primary thin-
ning: one, to retain this membranous characteristic as do both
the dorsal and lateral walls of the normal or albinous larvae
and the pituitary wall of the albino; the other, to develop into

44 According to Atwell, this lobe becomes recognizable in an 18 to 20 mm. stage
in R. pipiens.

MEMOIR NO. 11,

82 PIGMENTARY GROWTH AFTER ABLATION OF

the thickened wall characteristic of the pituitary floor of the
normal specimens. If freed from the influence of its buccal
associate and left to its own independent development, the
membranous structure persists; under the stimulatory influence
of its buccal associate, a thickening results, leading to the solid,
firm structure of the normal tadpole. Curiously, in the absence
of the buccal hypophysis, the inherent capacity for growth
expresses itself in the neural lobe as if an hereditary influence
here exerts itself and leads to the formation of a ‘vestigial’
structure. That the epithelial hypophysis does exert a stimu-
latory effect upon the adjacent neural tissue seems clear, as
has been pointed out, not only from the evidence accruing from
a study of the albino in its different stages, but from the ‘partial’
albino as well. The diminutive and misplaced fragment of the
buccal hypophysis in these specimens has invariably formed
about itself a brain lobe, identical in structure, so far as can be
determined, with the normally placed pars nervosa of the pitui-
tary. Moreover, the ‘influence’ of this vestigial epithelial gland
appears to be effective at some distance, as indicated by certain
specimens which, in sharp contrast to the albino, have no apic-
ally placed neural lobe. This condition suggests that this lobe
has either been inhibited and a new atypically placed lobe formed
de novo or it has been attracted to a new position by this mis-
placed epithelial fragment.

The inherent capacity for growth existing in the three por-
tions of the infundibular process and the response of these por-
tions to the presence or absence of the buccal component of the
hypophysis can perhaps best be shown by schematic curves
illustrating the reaction of the pituitary floor and one of the
other portions of the infundibular process under the two con-
ditions of normality and buccal hypophysectomy (fig. 10).
These curves show the development of the pituitary wall in a
normal tadpole and its albinous mate, together with a third
curve showing the development of the dorsal and lateral walls
of the infundibular process in either normal or albinous tad-
poles,

THE PARS BUCCALIS OF THE HYPOPHYSIS 83

Early in this section of the paper we stated that two lines
of evidence indicated that the neural lobe is dependent upon
its epithelial companion for its complete development. These
briefly are: 1) certain cellular elements from the pars epithelialis
(pars intermedia) apparently migrate into the neural component
of the hypophysis and contribute to its secretion; 2) by appro-
priate chemical treatment a substance can be secured from the
anterior lobe which displays certain of the physiological char-
acteristics typical of extracts of the posterior lobe of the pituitary.
From this study a third line of evidence from the experimental
and developmental work herein detailed has been secured; 3)
the neural lobe and pituitary floor are dependent upon the
association with the epithelial hypophysis for their full develop-
ment. The epithelial hypophysis then appears to exert a stimu-
lating effect upon these structures and even upon any adjacent
neural tissue, as is shown by the hypertrophy of the neural
tissue about the atypically placed epithelial lobe.

The thyroid

Sections of the larger albinous larvae show that the thyroid
glands of these specimens have suffered a profound atrophy,
an atrophy so intense that the remaining vestige could hardly
be suspected of playing any important physiological réle. That,
in fact, this is the case has recently been shown by Allen, whose
experimental animals, suffering a double extirpation of the
thyroid and buccal portion of the hypophysis, do not differ from
those deprived only of the epithelial hypophysis. Indeed, the
interrelationship obtaining between the hypophysis and thyroid
appears to be of such an intimate nature that the ablation of
the former, even in the midlarval stages, effects an atrophy of
the latter (Adler, 714).

The thyroid gland, then, is greatly diminished in size (table 7,
figs. 31 to 34) and modified in structure in those tadpoles suffer-
ing from an early and complete loss of the epithelial hypophysis.
By this atrophy the thyroid is diminished, in tadpoles in excess
of 36 mm. total length, to one-sixth or less of its normal size.

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THE PARS BUCCALIS OF THE HYPOPHYSIS 87

Structurally, this atrophy is expressed by a reduction in the
size of the follicles and in their colloid content (figs. 33 to 36).
Indeed, certain cell clusters would not be recognizable as a
follicular remnant, save for their location, so atypical are they.
Other follicles exhibit the characteristic organization and may
even contain an insignificant amount of poorly staining colloid.
In no case do we see the large-sized, colloid-filled follicle of the
normal animal. The component cells of the follicles appear also
to have suffered from the hypophysial deprivation. Their
cytoplasmic content is very noticeably diminished. This reduc-
tion in the size of the component follicles, together with an
apparent disappearance of some of them, results in a loose
organization of the gland, the majority of the follicles being
separated by wide intervals. Had these interfollicular spaces
been excluded in the models, an even greater discrepancy be-
tween the size of the thyroids of the albino as compared to the
normal would have been shown.

Although not evident in specimens of 10 mm. total length
(eighteen days after the operative stage), by the time larvae
have reached a length of 17 to 18 mm. (twenty-nine days after
the operative stage) the thyroid of the albinos is slightly but
invariably smaller than that of the controls. This discrepancy
progressively increases and is strikingly expressed in a 24-mm.
(thirty-nine days after the operative stage) albino as shown in
table 7. The pronounced atrophy of this gland in the albino
is not due, then, to a regressive process in the normal gland;
it is a phenomenon of retarded and atypical development.

It will be recalled that the ‘partial’ albinos, because of the
position assumed by the atypical epithelial fragment of the
hypophysis, were grouped into three classes: 1) those in which
there was no contact between the true neural lobe and the epi-
thelial fragment; 2) those in which there was a definite but
slight juxtaposition between these two components; and, 3)
those in which the epithelial fragment though diminished in size
nevertheless displayed its usual division into glandular and inter-
mediate lobes, which in turn displayed the usual relation with
the infundibular derivatives. Curiously, the thyroid glands of

88 PIGMENTARY GROWTH AFTER ABLATION OF

none of the animals included in these three classes are atrophic,
though the epithelial hypophysis be but one-sixth of its normal
size; indeed, those included in the second class exhibit greatly
hypertrophied thyroids with the frequent formation of accessory
glands (table 7, p 7, p 9, p 10). It will further be recalled
that only the specimens of the second and third classes com-
pleted metamorphosis, while those of the first class underwent
an abrupt and persistent metamorphic stasis at some stage in
this process. It would thus appear that by the slight reaction
between the true neural and the buccal components of the hy-
pophysis, a sufficient ‘hormonal’ substance was supplied to cause
an hypertrophic response of the thyroid gland.‘® These tad-
poles metamorphosed (class 2, specimens p 7, p 9, p 10). On
the other hand, if a larger amount of secretion was supplied
by the pituitary (class 3, specimen p 8), no hypertrophy of the
thyroid then resulted. A tentative explanation of the correla-.
tions obtaining between the epithelial and neural lobes of the
hypophysis and the thyroid in the first class of animals, those
in which no contact is made between the true neural lobe and the
epithelial fragment, is more difficult. Since the thyroid is of
normal size in these animals, it is evident that no compensatory
response of this gland was evoked, although there would seem
to be a need for such a response, since these animals did not
complete metamorphosis. It would appear that some inter-
action between the true neural lobe and the epithelial lobe is
necessary in order to evoke a hypertrophic response of the thy-
roid. Thus a functional specificity attends this union of true
neural lobe tissue with the reduced epithelial component of the
gland. The thyroid does not hypertrophy and metamorphosis
appears never to be completed when the epithelial hypophysis
comes in contact with only an atypical or a new neural lobe.

46 Tt is of interest to note in this connection that the thyroids, which were sub-
jected to microscopic examination, in Cushing’s canine hypophysectomies, ex-

hibited an excess of colloid. Further, that partial hypophysial extirpations in two
human patients resulted in thyroid enlargement (Exner, cit. after Cushing).

THE PARS BUCCALIS OF THE HYPOPHYSIS 89

The conditions then obtaining in these three classes of tad-
poles seem to indicate that the epithelial fragment where sep-
arated from the true neural lobe, either in itself or through its
reaction with brain tissue, other than the true neural lobe, is
able to stimulate the thyroid to normal development, but that
under these conditions metamorphosis cannot be completed;
that when this‘fragment is in very slight contact with the neural
lobe an hypertrophied thyroid results and complete metamor-
phosis is effected, and that when the usual relations of a com-
pleter contact between the two parts of the hypophysis obtain
no hypertrophy of the thyroid occurs and we have the comple-
tion of the metamorphic processes. It might thus appear that
the activity of the thyroid is thus the sole or determining factor
on which the metamorphic processes are dependent. That such
may not be the case seems clear from evidence gained by thyroid
feeding. In thyroid and thyroxin feeding extending for periods
in excess of six weeks and during which time daily minute doses
of thyroid or thyroxin were administered, I have not been able
to bring to complete metamorphosis a typical hypophysis-free
albinous tadpole. The early stages in this process were passed
through (leg growth, partial tail absorption), but the tadpole
could not be carried past a certain stage, death invariably super-
vening. This appears to be the case also with the ‘partial’
albinos of class 1, whose metamorphic processes could not be
carried to completion nor materially advanced after the onset
of the metamorphic stasis by instituting a carefully controlled
thyroid dosage. The results secured from the feeding of thyroid
to completely and partially hypophysectomized tadpoles stand
in sharp contrast to similar feeding experiments with the thy-
roidectomized tadpoles, which were readily completely meta-
morphosed by this treatment. It is not to be denied that a
more carefully regulated or a different dosage of thyroid might
bring about complete metamorphosis in the albino, but that it
would do so appears improbable from the evidence at hand.
The evidence thus indicates that metamorphosis is dependent
upon the active principle of at least two glands; in the absence
of either, the larval condition persists. One gland, the thyroid,

90 PIGMENTARY GROWTH AFTER ABLATION OF

can apparently initiate the process, but unless there be some
hypophysial secretion (and it would appear that this must be
formed at least in part by the interaction of the true neural
lobe with the epithelial component) the process apparently
cannot be completed.

The adrenal cortex and medulla

If the abdomen of a normal tadpole in the later larval stages
be opened and the ventral surface of the mesonephros be exam-
ined under the binocular, there will be seen a longitudinally
placed whitish-appearing cord. This cord, irregular in outline,
extends from a point somewhat cephalad to the kidney, over the
anterior four-fifths of this organ (fig. 37). This is the adrenal
gland.

When such a specimen is dropped into an alcoholic solution
of sudan ITI or scarlet R, this cord takes on a reddish hue. Sub-
sequent treatment with potassium bichromate reveals, however,
that not all the cells of this column are tinted with the fat dye,
for with chromation, groups of browned cells surrounded by the
lipoid-containing cords are revealed. Similar differential stain-
ing of the cortex is shown by osmium vapor or when an osmic-
bichromate solution is used (figs. 38 to 41). If potassium bich-
romate alone is used, however, it can be readily seen that only
the centrally placed cells of this strand give the chromaffin reac-
tion, a reaction participated in also by certain groups of cells
surrounding the central intestinal artery and neighboring parts
of the aorta. By these staining methods we may thus clearly
show the two components of the adrenal gland, the medulla
exhibiting the chromaffin reaction and the cortex; or interrenal
component, characterized by its lipoid content. The arrange-
ment of these two components resembles with surprising accuracy
that of the adult as described by Stilling (98) and Grynfeltt (’04).

The adrenal components of a thyroidectomized tadpole do
not differ in any marked degree from those of the normal animal,
yet there appears to be an increase in the cortical or interrenal
tissue not entirely explained by the larger size reached by these
animals (table 9).

THE PARS BUCCALIS OF THE HYPOPHYSIS 91

Albinous tadpoles, on the contrary, reveal notable variations
even on surface view from the picture presented by the two
normally pigmented types (fig. 39). Although the kidney is of
normal size, the adrenal column appears much more slender and
more mesially placed than in a normal or thyroidectomized
specimen of corresponding or even of inferior size. Not only
is the column more delicate, but its caudal extent is somewhat
less, its cephalic extent, however, corresponding to the normal.
Sudan III does not color the column deeply, while the osmic-
bichromate solution produces only a grayish coloration in con-
trast to the dark brown of the normal gland. Treatment with
potassium bichromate gives an effect nearly identical with that
of the normal. We thus have evidence, from surface views alone,
which leads us to suspect that the adrenal cortex of the albino
is diminished.

A section study of the three above-described types of animals
lends corroborative evidence of an unquestioned nature to that
furnished by the surface examination. For such a study it is
essential that the technique employed not only preserve the
lipoid content of the cortical or interrenal cells, but that the
medulla be clearly, although not necessarily differentially, stained.
The most satisfactory fixing fluid has proved to be an osmic-
bichromate mixture (Flemming’s fluid less the acetic acid) which,
although blackening the lipoids, does not interfere with the
subsequent staining of the other tissues. Curiously, the chro-
maffin reaction is not shown when the osmium and bichromate,
either mixed together or subsequent to each other, are used.
Following fixation, immersion in 50 per cent. alcohol for twenty-
four hours deepens the color of the lipoid granules and appears
to render them less soluble in the higher alcohols and the clear-
ing agents. The sections (5 to 8) are rapidly run down to
60 per cent. alcohol and then stained for two to three hours in
Babes’ safranin at 36°. After rinsing in alcohol, rapidly de-
hydrating and mounting, there is presented a preparation in
which the lipoid granules, a few of which have apparently been
partially dissolved, are distinctly shown and in which the chro-
maffin cells can be unmistakably identified by their red tinge.

92 PIGMENTARY GROWTH AFTER ABLATION OF

Further evidence that this identification is not incorrect was
secured by fixation in Miiller’s fluid followed by a prolonged
mordantage in potassium bichromate, the sections then showing
the reticular fat-free cortical cells and the browned medulla.

By the employment of these two methods it can be readily
determined that this longitudinal cell mass, aside from blood-
vessels, connective tissue, and an occasional readily identified
nephric tubule, is composed entirely of adrenal cortex and
medulla.

The cortical cords, varying somewhat in width, are usually
formed of three or four cell columns. They measure in the
normal animal from 30 to 484 in diameter, an average of 36.6u.
In the thyroidectomized specimens they are from 24 to 56v.
in diameter, an average of 36.54 (table 8). The individual
cells composing these cords are of approximately the same size
in the two animals, in the normal averaging 15.1u in diameter,
in the thyroidectomized specimens 16.5y. These cells are com-
posed of a mass of lipoid granules imbedded in a cytoreticulum
and surrounding a nucleus approximately centrally placed. This
structure, then, after the osmic-bichromate solution gives a
coarse, dark granular appearance, or after ordinary fixation
and treatment with fat solvents reveals a delicate reticular cell.

If we compare these cortical cords, or cells, of the albino with
those of the unoperated tadpole (figs. 38 to 41), we find that
they are decreased in size in the former. The cords (in a 55-mm.
albino) vary from 24 to 32u in diameter, an average of 23.7u,,
a reduction of approximately 33 per cent. from the normal.
The cells vary from 12 to 16% in diameter and average 12.7.
(table 8), a reduction of approximately 25 per cent. from the
normal. It is difficult to determine, because of their profuse
branching, whether the cords are actually reduced in number in
the albino, but apparently this is the case.

The interrenal cells of the normal or thyroidless animal are
browned by osmic; those of the albino assume a gray tone (figs.
38, 39). This would appear to be due to a different reaction
of these granules and not to their volume, since the granules of
both. types of specimen are blackened by subsequent treatment
with a low grade of alcohol.

93

THE PARS BUCCALIS OF THE HYPOPHYSIS

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94 PIGMENTARY GROWTH AFTER ABLATION OF

In order to determine accurately the diminution in the adrenal
cortex in the albino and its increase in the thyroidectomized
tadpole, this substance was accurately drawn with the camera
at a magnification of 300 diameters in three specimens, a 67-mm.
albino, a 56-mm. thyroidless tadpole, and a 42-mm. normal
animal. The drawings were transferred to a wax plate of pro-
portionate thickness and the weight of the models of the cortex

TABLE 9
Table giving the weight of wax models (800) of cortex of left adrenal (frog tadpoles)
Weight of
SPECIMEN model in
grams
Age, dated
Individual from opera-
specimen Type Length! tive stage Diet
number (days)
36 Albino 67 228 Anterior lobe 144.43
-/-/0.2 May 6 to July 18;
then liver
41 Normal 42 228 Liver 382.75
-/-/12
t 2 Thyroidec- | 56 228 Anterior lobe 759.60
tomized |-/-/2.0
p ll Partial 41 286 Same as spec. 36 360.15
albino -/-/16

1 The length of the component parts of the tadpole is given in the following order:
Total /Hind/Fore
Body/Tail/legs /legs

thus secured for these three specimens (table 9). The cortical
substance of one adrenal—the left in each case—was treated in
this way The albino, although 11 mm. longer than the thy-
roidectomized tadpole, and 25 mm. longer than the normal speci-
men, had but 38 per cent. of the cortical tissue presented by the
normal and 19 per cent. of that of the thyroidectomized speci-
men. It would thus appear to be established that the cortical
tissue is greatly diminished in the albino as compared with

THE PARS BUCCALIS OF THE HYPOPHYSIS 95

the normal or thyroidectomized tadpole. On the other hand,
the cortical adrenal tissue of the thyroidectomized tadpole
exceeded by twice that of the normal. Although the thyroid-
less animal is somewhat larger than the normal (11 mm.), yet
the discrepancy in the size of the adrenals is out of proportion
to the difference in the size of the specimens, and it thus seems
certain that the cortical tissue is hypertrophied in the thyroid-
less larva as compared to the normal.

Changes as striking but of a different nature are evident
between the adrenal medulla of a normal or thyroidectomized
tadpole and a hypophysectomized tadpole. In the normally
pigmented larva the medullary cells present a diverse appear-
ance, since certain cells are deeply stained and the cytoplasm
limited by a definite cell membrane, while the opposite extreme
is seen in certain other cells whose cytoplasm is reticular or even
vacuolated and but slightly tinged by the dye, the cell membrane
not being distinguishable (fig. 40). Between these two extremes
all gradations can be found, a condition suggestive of the various
secretory states exhibited by the mammalian chromaffin tissue.
In the albino, on the other hand, the cells are of one type; their
cell boundaries are distinct and their cytoplasm moderately and
uniformly stained (fig. 41). Moreover, the cells are uniformly
larger than those either of the normal or thyroidectomized tad-
pole as shown by table 8.

Not only are the chromaffin cells larger in the albino, but
their relation to each other and to the surrounding tissue is
peculiar to these animals. They closely approximate each other
and the adjacent cortical tissue, spaces between or around them
being seldom evident. In the normal or thyroidectomized tad-
pole, on the other hand, an interval frequently separates these
cells from each other and from the cortical tissue. This con-
dition, which simulates shrinkage, was at first believed to be
an artifact. Its constant occurrence in the normal and absence
in the albino with identical technique suggests two possible
causative factors. Either these spaces exist during life or the
physical constitution of the cells of the normal and thyroidless
larvae is such that shrinkage inevitably occurs during the manipu-

96 PIGMENTARY GROWTH AFTER ABLATION OF

lations involved in the technical treatment. Be the explanation
as it may, it is certain that in the preparations the chromaffin
tissue of the normal animal almost invariably does not fill the
intracortical space accorded it.

A duplication in wax of the volume of the medulla (x 187.5)
has been made for each of the three types of animal. In the
normal and thyroidectomized animals the total intracortical
medullary space has been included. It is evident, then, that if

TABLE 10
Table giving the weight of wax models (X187.5) of adrenal medulla (frog tadpoles)
Weight of
SPECIMEN model in
grams
Age, dated | |
Individual - from opera-
specimen Type Length! tive stage Diet
number (days)
34 Albino 54 217 Anterior lobe 27.32
19/35/0.2 May 6 to July 18;
then liver
39 Normal 39 217 Liver 14.93
* 15/24/4.5
t3 Thyroidec- 55 217 Anterior lobe 24.46
tomized 19/36/1.0

1 The length of the component parts of the tadpole is given in the following order:
Total /Hind/Fore
Body/Tail/legs /legs

only the actual chromaffin tissue exclusive of the surrounding
spaces had been drawn, an undertaking too laborious and uncer-
tain to be considered, the mass of the model in the normal and
thyroidless larvae would have been considerably reduced. From
the included table it will be seen that the volume of the medulla
while not seriously out of proportion to the size of the specimens
would appear to be increased in both the albino and the thyroid-
less larvae (table 10).

Attention has already been repeatedly called to the incom-
pletely hypophysectomized tadpole, the so-called ‘partial’ albino,

THE PARS BUCCALIS OF THE HYPOPHYSIS 97

and their characteristics described. Especial interest is associ-
ated with these specimens in the study of the adrenal, since the
pigmentation in Addison’s disease is usually referred to a de-
rangement of the adrenal. Both whole mounts and sections,
however, reveal the fact that neither the adrenal cortex nor
medulla suffers a serious disturbance in the ‘partial’ albino as
compared with the normal animal. To test more exactly the
amount of cortex in one such specimen, it was reproduced in wax
(x300), as has been previously explained. These findings show
that the cortical tissue is present in relatively as great an amount
as in the normal tadpole (table 9). Apparently, then, as in the
case of the thyroid, a relatively small amount of hypophysial
tissue is sufficient to give rise to a normal adrenal.

The epithelial bodies

The epithelial bodies (Maurer) might be suspected of par-
ticipating in the general endocrine upset experienced by the
tadpole suffering from pituitary deficiency. Such, indeed, ap-
pears to be the case, although these bodies do not suffer as do
the thyroids, adrenal cortex, or neural hypophysis. Models of
these glands made from four normal and four albinous tadpoles
reveal in many cases a profound diminution in the albino, al-
though in other cases this decrease does not transcend the limits
of variation of the individual bodies in the normal. When we
take cognizance of the total amount of tissue, however, there
can be no question but that it is profoundly diminished in the
albino (table 11). There appears to be no serious structural
abnormalities in these bodies. Thus in their reaction to epi-
thelial hypophysectomy these bodies align themselves with all
the other endocrine organs thus far examined (thyroids, adrenal
cortex, neural hypophysis) save one, the adrenal medulla. An
opposite response—one of increasing size—is evoked in the
epithelial bodies (Allen) by thyroidectomy. In this opposed
response in thyroidectomy they thus align themselves with the
enlargement which the pituitary (Allen, Hoskins) and adrenal
cortex (Smith) enjoy.

MEMOIR NO. 11.

PIGMENTARY GROWTH AFTER ABLATION OF

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THE PARS BUCCALIS OF THE HYPOPHYSIS 99

The fat-organ

Attention is called to the fat-organ, a structure which serves
as a fat storehouse and as such is subject in the adult frog to
seasonal and nutritional variations (Gaupp), since the behavior
of this ‘organ’ in the tadpole suffering from hypophysial de-
ficiency strongly simulates the behavior of the adipose tissue
in the mammal suffering from hypopituitarism. The normal
tadpole completing metamorphosis retains but a vestige of the
former robust organ. Similarly, the fat-organ of a thyroidec-
tomized tadpole subjected to inanition, or metamorphosed by
thyroid administration, is greatly decreased in size. This is not
the case with the albino (fig. 60). After prolonged starvation
no decrease in the large size attained by this organ in thege
specimens is evident. In the ‘partial’ albino, too, although
the animal undergoes a prolonged metamorphic period, this
organ appears not to be reduced in size, though the nutritional
needs of such an animal must be extreme. This was well shown
by the two specimens of figure 53. The partial albino, the most
emaciated of the two, had an immense fat-organ; indeed, the
fat-organ and the kidneys were the major abdominal structures.
This ‘organ’ in the other specimen, however, a normal, was
scarcely recognizable. The large size and persistency of this
structure in the tadpoles suffering from a complete or partial
ablation of the epithelial component of the pituitary, appears
not to be unlike the adiposity exhibited by the mammal after
partial hypophysectomy (adipositas universalis, Cushing) or in
the human subject suffering from hypopituitarism. These ani-
mals thus furnish abundant testimony of the disturbance in fat
metabolism brought about by these conditions and indicate that
even under the extreme exigencies of inanition the utilization
of the fat is impossible.

100 PIGMENTARY GROWTH AFTER ABLATION OF
5. DISCUSSION

It has been possible to determine the contribution which
each group of pigment cells makes to the striking picture of
‘albinism’ produced by the operation of epithelial hypophy-
sectomy in the early larvae of the toad and frog. Evidence from
at least two sources (feeding experiments and epithelial ex-
changes) points to the importance of the paucity of the epidermal
melanin in the formation of this picture. By this paucity and
the consequent greater transparency of the epidermis the double
sheet of broadly expanded xantholeucophores are permitted to
display their full iridescent and metallic effect. The expansion
of the xantholeucophore group of cells is essential to this picture,
since with their contraction the albinous picture lost its char-
acteristic metallic appearance and the larvae became notably
darkened in color. Moreover, that the participation of the deep
melanophores in this picture was of no great significance was
evident not only from the masking which these cells suffer due
to the expanded ‘interference’ cells, but from a variety of experi-
mental procedures.

The atypical physiological condition of the pigment cells
contributing to this picture of albinism is unquestionably refer-
able to a fault in the endocrine system and not to an altered
nervous mechanism. Not only is evidence of an unequivocal
nature presented on this point as regards the altered physiological
state of the xantholeucophores and epidermal melanophores by
the skin exchanges, but the effect of endocrine extracts and
diets on the atypical pigmentary system lends adjuvant evi-
dence.

With the complex interrelationships obtaining in the internal
secretory system, however, some difficulty has been encountered
in referring the pigmentary disturbance exhibited by the tad-
poles suffering from hypophysial deficiency to a definite locus
in this system. Yet by the correlation of the evidence accruing
both from the structural pictures presented by the members of
this system together with the alterations in the pigmentary
system produced by various dietary régimes and by immersion

THE PARS BUCCALIS OF THE HYPOPHYSIS 101

of the albino in endocrine extracts, the specific endocrine de-
ficiency would appear to be disclosed. In this we have been
greatly aided by the different endocrine pictures obtaining in
completely and partially hypophysectomized larvae, for it will
be recalled that both of these types of animals exhibit a pig-
mentary fault. Since all the endocrine glands save the pituitary
are of normal structure in the partially hypophysectomized (but
albinous) larvae, it would appear that neither the adrenal com-
ponents, the epithelial bodies, nor the thyroid are at fault.
The thyroid can further be freed from participation in this
disturbance since the pigmentary system of the thyroidectomized
tadpole is normal. Further, it will be recalled that the feeding
of posterior-lobe tissue (including the pars intermedia), alone,
of all the administered glandular substances (thyroid, adrenal
cortex, adrenal medulla, and anterior lobe), effected a partial
replacement of the epidermal melanin, although, curiously, at
the same time, increasing the abnormal contraction of the epi-
dermal melanophores. It was further shown that the immersion
of these larvae in pars intermedia emulsion alone of the internal
secretory extracts produced a normal functional state in the
chromatophore system. Thus, the evidence derived from these
three lines of investigation all points to a fault in the posterior-
intermediate lobe secretory mechanism as being responsible for
the pigmentary disturbance obtaining in the larvae suffering
from either a partial or total loss of their epithelial hypophysis.

The alimentary assimilation of the fresh glandular lobe of
the pituitary has supplied the growth-maintaining principle to
the animals exhibiting a retarded growth rate induced by buccal
hypophysectomy.‘® Unequivocal proof of this is furnished by

46 What element of this structurally diverse gland contributes this growth prin-
ciple is obscure. Histological examination of frozen sections (15 to 20) reveals
that extraction with boiling absolute alcohol does not appreciably diminish either
the number or size of the most characteristic element of this gland, the acidophilic
granulations. Evidence determinative of the dissolving action of boiling distilled
water is more uncertain because of the macerating action upon the connective
tissues and the consequent difficulty in the examination of this material. Yet it is
certain that a considerable number of these granules survive such treatment. That

the tissue thus extracted exhibits the characteristic growth effects of the fresh gland,
while the extracts do not exhibit such effects, supplies evidence against these granu-

102 PIGMENTARY GROWTH AFTER ABLATION OF

the frog, supportive evidence by the toad tadpole. Extraction
of this anterior-lobe substance with either boiling absolute
alcohol or boiling water appears not to remove this principle,
since the albinous tadpoles supplied with such extracts exhibit
the retarded growth rate typical of the liver-fed albino, while
those larvae supplied with the residues remaining after extrac-
tion grow at a normal rate.

We have pointed out the structural modifications in the various
organs of the internal secretory system resulting from the early
ablation of the pars epithelialis of the hypophysis. By these
alterations eloquent testimony as to the interdependence of the
various widely separated members of this correlative system has
been secured. It will be recalled that the response of these
organs to the complete loss of the epithelial hypophysis led to a
diminution in their size, save in one gland, namely, the adrenal
medulla. It was also pointed out that the opposite response
is evoked by the early removal of the thyroids, the members
of this system undergoing an increase in size, save again the
adrenal medulla, which appears not to be seriously altered. It
is thus seen that the response of the organs of internal secretion
(save the adrenal medulla) to epithelial hypophysectomy is in
the reverse direction to that resulting from thyroidectomy, the
loss of the hypophysis causing a diminution, the loss of the
thyroid an increase in their size.

It was further pointed out that the presence of a relatively
minute remnant of the epithelial pituitary was sufficient to
abort the structural anomalies in these organs arising from the
complete loss of the epithelial hypophysis, save the case of the
derivatives of the infundibular process. These derivatives, of
which the major member is the neural lobe, are always affected
in any partial epithelial hypophysectomy. This we were inclined
to refer to the close anatomical relationship obtaining between
these two components of the pituitary. For it was shown that

lations being merely a by-product or a refuse accumulated during the secretory
process. It must be admitted that such evidence is inconclusive, however, since
the part played by the other cells, the basophiles and chromophobes, whose cyto-
plasm appears to be more labile than that of the acidophiles, has not been excluded.

THE PARS BUCCALIS OF THE HYPOPHYSIS 103

in the case of the complete absence of the epithelial hypophysis
the neural lobe underwent an abortive development, while the
other portion of the infundibular process, the pituitary floor,
normally in contact with the epithelial component, retained its
membranous character in sharp contrast to the thickened wall
normally displayed. Further studies of the ‘partial’ albino indi-
cated that the atypically placed epithelial fragment apparently
is able to ‘stimulate’ the adjacent neural tissue to form a struc-
ture, simulating histologically the typical neural lobe. Doubt,
however, was cast upon the functional sufficiency of this ‘novel’
neural lobe, since the animals displaying this anatomical arrange-
ment failed to metamorphose in contrast to those in which this
epithelial fragment attained contact, though but slight, with
the true neural lobe, a failure not entirely attributable to the
smaller’ size of this epithelial remnant. From this it would
appear that the neural tissue comprising the pars nervosa of
the pituitary enjoys the same functional specificity as do the
other glandular tissues of the body.

It is a pleasure to acknowledge the aid which I have received
throughout this work from Doctor Evans. The many helpful
suggestions and the encouragement which he has given during
the progress of the work and in the preparation of the manuscript
have been invaluable. To Mrs. Smith and Mr. Lee for their
aid in the preparation of many of the models I am much in-
debted. Acknowledgment is made for the services rendered
by our photographer, Mr. Matthews, and by our artist, Mr.
Sweet.

6. SUMMARY

1. The frog and the toad tadpole, because of the accessi-
bility of the epithelial hypophysis in the early larval stages and
the prolonged survival of the animals suffering the loss of this
gland, has proved of great value in the institution of hypophysial
disturbances and in the subsequent analysis thereof.

104 PIGMENTARY GROWTH AFTER ABLATION OF

2. The early removal of the epithelial hypophysis induces a
striking disturbance, 1) in the pigmentary system, 2) in the
growth rate, and, 3) in the structural characteristics of most
of the other glands of internal secretion.

3. The pigmentary alterations resulting from early hypophy-
sectomy are expressed by, 1) a diminution in the epidermal free
pigment; 2) a diminution in the number and melanin content
of the epidermal melanophores, together with an abnormal state
of contraction; 3) a maximal.expansion of the xantholeucophores;
4) a partial contraction of the deep melanophores in the younger
larvae.

4. The first three above-mentioned alterations in this system
result in and are essential to the complete formation of the
picture of albinism; the fourth does not materially contribute to
this picture. This would be suspected from the anatomical
arrangement obtaining in these cell layers and can further be
proved by various experimental procedures by which each of
these factors can be modified: 1) the partial replacement of the
epidermal melanin (effected by posterior-lobe feeding) is seen to
blanket partially the underlying, broadly expanded xantho-
leucophores, the larvae being notably darker than their brothers
supplied with other diets; 2) the complete expansion or contrac-
tion of the deep melanophores of the albino effected by altering
the conditions of light and background does not materially
change the color of the albinous larvae, and conversely the con-
traction of these cells in the normal by the absence of light, while
productive of a translucency, does not produce an albino; 3) a
contraction of the xantholeucophores of the albino as exhibited
by an albinous graft to a normal host or by the use of a strong
anaesthetic not only results in their darkening, but in the loss
of the metallic silvery tone characteristic of the picture of al-
binism.

5. A developmental study of the pigmentary system of the
albino reveals the fact that the epidermal melanophores appear
at a later date and in diminished numbers as compared to the
normal; that the free epidermal melanin suffers a relatively
greater diminution than in the normal, and that the xantho-

THE PARS BUCCALIS OF THE HYPOPHYSIS 105

leucophores appear at the same time and in approximately the
same numbers as in the normal, but exhibit from their earliest
appearance a broad expansion as compared to the punctate
character of these cells in the unoperated tadpole.

6. The broad expansion of the xantholeucophores and con-
traction of the epidermal melanophores of the albino is not due
to an alteration in their nervous mechanism, but to the modified
tissue fluids which bathe them. This is proved by the reciprocal
skin exchanges. That this alteration in the tissue fluids is of
a hormonal nature appears probable not only from the known
potency of these substances, but also because of the extensive
modifications suffered by the elaborators of these substances in
the albino. There appears to be no ground for referring the
deficiency in the epidermal melanin to other than an internal
secretory origin.

7. Various physiological and pharmacological experiments on
the pigmentary system of older albinous and normal larvae
reveal the fact that, 1) the deep melanophores of the albino and
the normal tadpole react identically under all tests tried by the
author; 2) the xantholeucophores of the normal animal respond
to changes in environmental conditions, those of the albino, on
the other hand, maintain a refractory expansion under all sub-
lethal stimuli tried by the author, save that afforded by the pars
intermedia emulsion; 3) the epidermal melanophores of the
albino invariably expand when subjected to the condition of
‘light and heat.’ Those of the normal tadpole react in a more
variable manner, not infrequently exhibiting the opposite reac-
tion, contracting instead of expanding.

8. The thyroid and the adrenal cortex are strikingly dimin-
ished in size in the albino; the epithelial bodies suffer a lesser
though definite diminution; the adrenal medulla suffers no un-
questionable quantitative changes, but it appears not improbable
that it is slightly hypertrophied. Structurally, the adrenal
medulla is clearly altered in the albino, since its cells are all of
a uniformly staining large type, as compared to the variability
in size and the diversity in appearance exhibited by the adrenal
chromaffin cells of the normal.

106 PIGMENTARY GROWTH AFTER ABLATION OF

9. The posterior lobe is invariably diminished in size, asym-
metrical in shape, and atypical in position in the albino. More-
over, in the albino the floor of the infundibular process—pituitary
floor—normally in contact with the epithelial hypophysis, does
not undergo the secondary thickening which takes place in the
normal subsequent to the juxtaposition of the epithelial hy-
pophysis with this structure, but retains its membranous struc-
ture.

10. Attempts to remove the buccal hypophysis occasionally
leave a sufficiently large fragment of the epithelial hypophysis
in place for complete regeneration to occur. Other instances of
incomplete removal of the epithelial part of the gland produce
animals which are altered in a characteristic way. We have
termed these ‘partial’ albinos. As far as their appearance is
concerned, in most instances these animals are typical albinos
and the incomplete nature of the operation is only disclosed
later when a development of the limbs appears. Indeed, some
of these ‘partial’ albinos furnished the only instances of the
complete metamorphosis of albinous larvae. Our present knowl-
edge of the intimate relationship of the thyroid to metamor-
phosis would lead us to predict that the thyroid gland at least,
was not interfered with. As a matter of fact, anatomical study
of all of the ‘partial’ albinos showed that not only the thyroid,
but that all of the internal secretory glands, with one exception,
were undiminished in size or degree of development. Indeed, in
a limited number of those ‘partial’ albinos which completed
metamorphosis the thyroid was greatly hypertrophied (colloid
goiter) and frequently accessory thyroids were formed.

11. The neural lobe of the ‘partial’ albino is usually dimin-
ished in size and is invariably atypical in shape and position.

12. About the atypically placed epithelial fragment of the
hypophysis of the ‘partial’ albino there is formed a ‘novel’
neural lobe, a structure apparently formed not only by the
indentation of this epithelial fragment in the brain tissue, but
by an actual hypertrophy of the adjacent parts. Not infre-
quently in these specimens there is no recognizable neural lobe
such as invariably is exhibited by the typical albino.

THE PARS BUCCALIS OF THE HYPOPHYSIS 107

13. The evidence thus indicates that the neural lobe and
pituitary floor are dependent upon the epithelial hypophysis
for their complete development, and further that an atypically
placed epithelial hypophysis has the power to cause an hyper-
trophy of the adjacent neural tissue.

14. Since ‘partial’ albinos may be secured in which the pig-
mentary upset or albinism is nearly as profound as in those
larvae which have suffered a complete epithelial ablation, and
since in these instances none of the endocrine glands are gravely
impaired in development save the hypophysis, and sincé the
feeding of posterior lobe (plus pars intermedia), alone, of the
major internal secretory glands effects a partial replacement
of the epidermal melanin in the albino, and further since no
extract save that of the pars intermedia induces a normal physi-
ological state in the pigmentary system of this animal, it seems
justifiable to refer the endocrine fault responsible for the pig-
mentary disturbance to the posterior lobe or the posterior-
intermediate lobe secretory mechanism and to free the other
glands from responsibility.

15. The frog tadpole suffering from complete or incomplete
hypophysial deficiency exhibits even after a long period of
inanition or after a prolonged metamorphosis a fat-organ un-
diminished in size, in contrast to the small size of this organ in
similarly treated thyroidless tadpoles or in normal tadpoles
near the end of metamorphosis. The persistence of the fat-
organ in these tadpoles appears to simulate the adiposity ex-
hibited by the mammal suffering from hypophysial deficiency.

16. The frog tadpole which has suffered the early loss of
its epithelial hypophysis exhibits a retardation in growth when
supplied with a continuous diet of liver, posterior lobe, adrenal
cortex, or adrenal medulla. This growth retardation is feebly
expressed at first, but becomes very pronounced at about the
midlarval period.

17. A continuous diet of the fresh anterior lobe of the beef
replaces the growth-‘maintaining’ substance lost by buccal hy-
pophysectomy and so effects a nearly normal rate of growth
in the albinous frog tadpole. Since these animals do not meta-

108 PIGMENTARY GROWTH AFTER ABLATION OF

morphose, their growth extends beyond the normal larval period.
Consequently they attain a size in excess of the normal.

18. The sensitiveness of the hypophysectomized frog tadpole
to the growth-‘maintaining’ substance of the anterior lobe makes
it possible by feeding experiments to test in a particularly effica-
cious manner any hypophysial substance for the presence or
absence of this principle.

20. Neither aqueous nor alcoholic extracts of the anterior
lobe of the pituitary nor the intraglandular colloid of the hy-
pophysis contain the growth-‘maintaining’ substance of this
gland. Conversely, the residues formed by extracting the
hypophysis with boiling water or boiling absolute alcohol con-
tain this growth substance.

THE PARS BUCCALIS OF THE HYPOPHYSIS 109

BIBLIOGRAPHY

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110 PIGMENTARY GROWTH AFTER ABLATION OF

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1918 Further experiments with thyroidectomy in amphibia. Proc.
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1916 The reactions of the melanophores of Amblystoma larvae.

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THE PARS BUCCALIS OF THE HYPOPHYSIS 111

Laurens, H. 1917 The reactions of the melanophores of Amblystoma tigrinum
larvae to light and darkness. Jour. Exp. Zodl., vol. 23, pp. 195-205.

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Lissen, S. 1906 Uber die Wirkung von Extrakten chromaffinen Gewebes (Ad-
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108-117.

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1918 The physiology of the melanophores of the horned toad
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vol. 37, pp. 393-426.

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1916 The effect of hypophysectomy in the early embryo upon the
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112 PIGMENTARY GROWTH AFTER ABLATION OF

Smiru, P. E. 1916 The effect of hypophysectomy upon the subsequent growth
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stracts, p. 94.
1918 The growth of normal and hypophysectomized tadpoles as
influenced by endocrine diets. Univ. Cal. Pub. in Physiol., vol. 5,
pp. 11-22.
1919 The pigment changes in frog larvae deprived of the epithelial
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1919 On the reaction of the pigment cells in normal and albinous
frog larvae. Ibid., pp. 78-80.
1919 Upon the experimental exchange of skin transplants between
normal and albinous larvae. Ibid., vol. 16, pp. 80-81.
1919 On the effects of ablation of the epithelial hypophysis on the
other endocrine glands. Ibid., vol. 16, pp. 81-82.

Sparta, R. A. 1913 Evidence proving the melanophore to be a disguised type
of smooth muscle cell. Jour. Exp. Zodl., vol. 20, pp. 193-215.
1916 The responses of single melanophores to electrical stimulation.
Am. Jour. Physiol., vol. 41, pp. 577-596.
1918 Concerning a new method of biological standardization of
pituitary extract and other drugs. Jour. Phar. and Exp. Ther., vol. 11,
pp. 209-219.

Srpinko, O. V. 1900 Bau und Entwickelung der Nebenniere bei Anuren. Anat.
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SrenDELL, W. 1913 Zur vergleichenden Anatomie und Histologie der Hypophysis
cerebri. Arch. f. mikr. Anat., Bd. 82, S. 289-332.

Stitytinc, H. 1898 Zur Anatomie der Nebennieren. Arch. f. mikr. Anat., Bd. 52,
8. 176-195.

Stumpr 1911 Zur Histologie der Neurohypophyse. Virchows Arch., Bd. 206,
8. 70-79.

Terry, G. 8. 1918 Effects of the extirpation of the thyroid gland upon ossi-
fication in Rana pipiens. Jour. Exp. Zoél., vol. 24, pp. 567-587.

Titnny, F. 1913 An analysis of the juxta-neural epithelial portion of the hy-
pophysis cerebri, with an embryological and histological account of.
a hitherto undescribed part of the organ. Intern. Monat. f. Anat. u.
Physiol., Bd. 30, 8. 258-293.

TRAUTMANN, A. 1909 Anatomie und Histologie der Hypophysis cerebri einiger
Sduger. Arch. f. mikr. Anat., Bd. 74, 8. 311-367.

VoceL, M. 1912 Das Pigment des Hinterlappens der menschlichen Hypophyse.

/ Frankfurt. Zeitschr. f. Pathol., Bd. 11, S. 166-191.

Weict, R. 1913 Uber homéoplastische und heteroplastische Hauttransplantation
bei Amphibien mit besonderer Beriicksichtigung der Metamorphose.
Arch. f. Entw.-mech. d. Organ., Bd. 36, 8. 595-625. :

WINKLER, F. 1910 Beobachtungen iiber die Bewegungen der Pigmentzellen.
Arch. f. Dermat. u. Syphilis, Bd. 100, S. 255-260.

PLATES

118

MEMOIR NO, 11.

ABBREVIATIONS

a.o., adhesive organ op.v., optic vesicle
hyp., hypophysis ol.v., otic vesicle
hyp.p., hypophysial pit pJf., pituitary floor
inf., infundibulum p.g., pars glandularis of the hypophysis
n.p.ep., vestigial epithelial lobe (pars epi- p.i., pars intermedia of the hypophysis

thelialis) p.n., pars neuralis of the hypophysis
n.p.n., new neural lobe i.p.f. thickened portion of pituitary oor
o.p., olfactory placode I, IT, ITT, branchial arches

PLATE 1

EXPLANATION OF FIGURES

11 A 4mm. frog larva (R. boylei) showing the surface characteristics at a favor-
able stage for epithelial hypophysectomy. (a) Ventrolateral, (b) ventrocephalic
view.

12 A median sagittal section of a frog larva of approximately the same age and
size as shown in figure 11.

114

PLATE 1

in 4 ay
(a e 3 Op.v. Fs op
(a) | : ; -otr ——hyp. p.

11

PLATE 2
EXPLANATION OF FIGURES

13 The pigmentary system” of a normal 43/9.0 “frog tadpole, ‘standard’ environ-
ment, showing the successive layers of free pigment and chromatophores.49 The
animal was fixed by dropping into Helly’s fluid 101 days after the operative stage.
Liver diet. 226.

14 The pigmentary system of a 43/0.1 albino frog tadpole, ‘standard’ environ-
ment. Fixed by dropping into Helly’s fluid 101 days after epithelial hypophy-
sectomy. Liver diet. 226.

15 The pigmentary system of a 10-mm. (total length) normal frog tadpole,
‘standard’ environment. Dropped into Helly’s fluid 22 days after the operative
stage. Liver diet. The epidermal melanophores are just appearing. 226.

47 All the drawings of the pigment cells (figs. 13 to 23) were made from skin whole
mounts taken from the dorsal region of the body.

‘8 The total length of the specimen is shown by the first figure, the hind-leg
length by the second.

“In the pigment cell drawings the most superficial layer is shown on the left,
the deepest on the right. From left to right they are in order, (a) the layer of free
epidermal pigment, (b) the layer of epidermal melanophores, (c) the xantholeuco-
phores, (d) the deep melanophores.

116

PLATE

15

Re. TUPLE TG P=

9

PLATE 3
EXPLANATION OF FIGURES

16 The pigmentary system of a 10-mm. (total length) albino frog tadpole, ‘stand-
ard’ environment. Dropped into Helly’s fluid 22 days after the operative stage.
Liver diet. The epidermal melanophores have not yet formed. 226.

17 The pigmentary system of a 14.5-mm. (total length) normal frog tadpole,
‘standard’ environment. Fixed by dropping into Helly’s fluid 47 days after the
operative stage. Liver diet. 226.

18 The pigmentary system of a 14.5-mm. (total length) albino frog tadpole,
‘standard’ environment. Fixed by dropping into Helly’s fluid 47 days after the
operative stage. Liver diet. 226.

PLATE 3

Pe Suc 709

119

PLATE 4
EXPLANATION OF FIGURES

19 The pigmentary system of a ‘light and heat’ adapted 38/6.0 normal frog
tadpole which exhibited a contraction of the epidermal melanophores under the
influence of this stimulus. Fixed 514 months after the operative stage by dropping
into Helly’s fluid. Liver diet. 226.

20 The pigmentary system of a ‘light and heat’ adapted 46/0.5 albino frog
tadpole. Fixed 514 months after epithelial hypophysectomy by dropping into
Helly’s fluid. Liver diet. 226.

21 The pigmentary system of a 46/0.1 albino frog tadpole, supplied with a
continuous diet of posterior lobe, ‘standard’ environment. Fixed by dropping into
Helly’s fluid, 414 months after epithelial hypophysectomy. 226.

120

PLAT HO

20

RS we 109

PLATE 5
EXPLANATION OF FIGURES

22 The pigmentary system of a 46/0.1 albino frog tadpole, ‘light and heat’
adapted. This tadpole was supplied with a posterior-lobe diet for 414 months.
Prior to fixation it had been on a liver diet for one week. Fixed 444 months after
epithelial hypophysectomy. 226.

23 The pigmentary system of a 43/0.1 albino frog tadpole, ‘light and heat’
adapted. This tadpole was supplied with a posterior-lobe diet for 3144 months.
It had been on a liver diet for 5 weeks prior to fixation. Fixed by dropping into
Helly’s fluid 414 months after epithelial hypophysectomy. 226.

PLATE 5

MERE a 22
4 a
f a ee

123

PLATE 6
EXPLANATION OF FIGURES

24 A diagram of the ventral view of the brain of a 45/6.0 frog tadpole to show
the position of the hypophysis.

25 A model of the infundibular process and hypophysial components of a 38/3.5
normal frog tadpole (specimen 15), fixed 63 days after the operative stage. Liver
diet. The caudal end of the infundibular process faces the top of the page. u,
dorsal, 6, ventral, c, median sagittal, views. X89.

124

PLATE 6

PLATE 7
EXPLANATION OF FIGURES

26 A model of the infundibular process and hypophysial components of a 43/0.1
albino frog tadpole (specimen 18), fixed 101 days after epithelial hypophysectomy.
Liver diet. The caudal end of the infundibular process faces the top of the page.
a, dorsal, b, ventral, c, median sagittal, views. X89.

27 A model of the infundibular process and hypophysial components of a 36/4.2
‘partial’ albino frog tadpole (specimen, p 1), fixed 66 days after the operative stage.
Liver diet. The caudal end of the infundibular process faces the top of the page.
a, dorsal, b, ventral, c, median sagittal, views. X89.

126

PLATE 7

PLATE 8
EXPLANATION OF FIGURES

28 A median sagittal section through the infundibular process and the hypo-
physial components of a 38/3.5 normal frog tadpole. Liver diet. Fixed 63 days
after the operative stage. 227.

29 A median sagittal section through the infundibular process and the hypo-
physial components of a 43/0.1 albino frog tadpole (specimen 16). Liver diet.
Fixed 101 days after epithelial hypophysectomy. 227.

30 A median sagittal section through the infundibular process and the hypo-
physial components of a 36/4.2 ‘partial’ albino frog tadpole (specimen p 1). Liver
diet. Fixed 66 days after the attempted epithelial hypophysectomy. 227.

128

129

MEMOIR NO. 11.

PLATE 9
EXPLANATION OF FIGURES

31 (a) Ventral and (b) median views of a model of the left thyroid of a 38/3.5
normal frog tadpole (specimen 15). Liver diet. Fixed 63 days after the operative
stage. X89.

32 (a) Ventral and (b) median views of a model of the left thyroid of a 43/0.1
albino frog tadpole (specimen 18). Liver diet. Fixed 101 days after epithelial
hypophysectomy. X89.

130

PLATE 9

151

PLATE 10
EXPLANATION OF FIGURES
33 A cross-section through the largest portion of the left thyroid of a 40/5.0

normal frog tadpole. Liver diet. Fixed 64 days after the operative stage. 227,

34 A cross-section through the largest portion of the left thyroid of a 40/1.5
albino frog tadpole. Liver diet. Fixed 64 days after epithelial hypophysectomy.
X 227.

35 The follicle, shown by an arrow, in figure 33. 765.
36 The follicle, shown by an arrow, in figure 34. 765.

PLATE 10

PLATE 11
EXPLANATION OF FIGURES
37 The ventral surface of the dorsal abdominal wall of a 44/9.0 frog tadpole to

show the position of the adrenals.

38 The adrenal cortex of a 43/8.0 normal frog tadpole. Drawn from a whole
mount. Osmium-bicromate fixation. The specimen was killed 221 days after the
operative stage. Liver diet. 24.

39 The adrenal cortex of a 55/0.2 albinous frog tadpole. Same fixation, age,
and diet as above. X24.

134

PLATE tt

PLATE 12
EXPLANATION OF FIGURES

40 A small portion of the adrenal of a 48/9.0 normal frog tadpole. Liver diet.
Fixed in osmium-bichromate 195 days after the operative stage. Babes’ safranin
stain. Cortex black, medulla and nuclei red. 733.

41 A small portion of the adrenal of a 54/0.2 albino frog tadpole. Same age,
diet, fixation, and stain as above. 733.

136

PLATE 12

41

40

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PLATE 17
EXPLANATION OF FIGUItES

55. Photographs of a normal and an albinous frog tadpole to show the xantho-
leucophores. Photographed May 26th. The same specimens as shown in figure 44.
x7.

56. An enlargement of a portion of the dorsal body surface of the specimens
shown in figure 55.

146

PLATE 17

56

55

147

PLATE 18
EXPLANATION OF FIGURES

57. An albinous and a normal frog tadpole showing reciprocal skin exchanges.
Taken four hours after the skin exchange was effected. Age of specimens 70 days,
dated from the operative stage. 5.

58. Enlargements of the skin exchanges and surrounding region of the specimens
shown in figure 57.

148

PLATE 18

OT

149

PLATE 19
EXPLANATION OF FIGURES

59. The mesonephroi and adrenal bodies, fixed in the osmium-bichromate solu-
tion. (a) ‘Partial’ albino young adult frog (specimen p. 7). (b) Normal young
adult frog (specimen 33). (c) Albinous frog larva. Age of specimens 356 days.
Specimens a and b are shown in figure 53.

60. An albinous (a) and a thyroidless (b) frog tadpole photographed to show the
fat-bodies. Taken immediately after death. These specimens had been subjected
to inanition for six weeks prior to death. Age 335 days. 2.

150

PLATE 19

60

ao

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