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thesis entitled

The liistoloLg of the Anterior
Pituitary, Thyroid and Adrenal of
Thyroid-stimulated Purebred
English Bulldogs
presented by

Esther M. Smith

has been accepted towards fulfillment
of the requirements for

Major professor

Date‘ ' 97//5/

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This is to certify that the
thesis entitled

The distolog; of tne Anterior
Pitiitary, Thyroid and Adrenal of
ThsToid-stirmlatcd First-rod
English bulldogs
presented by

Esther H. Smith

has been accepted towards fulfillment
of the requirements for

‘7 f‘
l

' o .' L, -
_i_degree m Anatomy

67/ <d7. I LAC/flux

Major professor

 

Date 22 2/17/17 A ’7 /; /105’./

I

 

 

 

Il‘lql.lltll Ll‘lLLll 1114 I'll] IIL

 

JE HISTOLOGY OF THE AKTLRIOR PITUITARY,
”YROID AID ADRELAL CF THYRCID-STIXULATED

EUREEHED EEGLISH BULLDOGS

T
r

4
A.

T
T

By-

Esther K. Smith

A Thesis

Submitted to the School of Graduate Studies of

Michigan State College of Agriculture and
Applied Science in partial fulfillment
of the requirements for the degree
of

MASTER OF SCIEKCE

Department of Anatomy
1951

THESlS

TABLE OF COETENTS

INTRODUCTION
REVIEW CF LITERATURE
Thyroid

Anterior Pituitary
Adrenal

KATERIALS AID KETHODS

RESULTS AED DISCUSSION

Thyroid
Anterior Pituitary
Adrenal

SUUIARY AHD CONCLUSIONS

LITERATURE CITED

35485?

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40

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ACKNOWLEDGMENTS

The author wishes to express her sincere ap-
preciation to Dr. M; Lois Calhoun under whose in-
spirational guidance and unfailing confidence, this
work was accomplished. Also to acknowledge indebtedness
to Dr. Paul Reineke of the Department of Physiology for
making these animals available to the author, and for

the many hours of consultation.
Many thanks are extended to Dr. Pauline Paul,

Department of Home Economics for her help with the
statistical analysis of the data; to Dr. Charles
Titkemeyer who did the post mortems on these animals; to
Dr. Frank Thorp, Jr. and Dr. Frederick Smithoors for their
helpful suggestions and assistance.

The writer wishes to thank Dean Armstrong, De-
partment of Conservation, for the excellent drafting Job
on the charts which made subsequent photography of then

a relatively‘simple matter.

IIIT‘WDUCTIOII

This problem was undertaken as a continuation
of a study which investigated the effect of exogenous
thyroid upon the frowth rate of purebred English bull-
dog puppies (borgman and fleineke l949).

Since histolOgists seldom have an opportunity
to study tissues obtained from purebred animals, and
inasmuch as the microscopic anatomy of the endocrine
orfans varies with the different breeds of dogs, it is
heped that this work will be of Value as a foundation
for fhrther study. According to S ockard (1941), no
other mammalian species differs so widely in structural
type as do the breeds of domestic does. The internal
organs and functional reactions among different breeds
show wide deviations from the known mongrel dog pattern.
Gilmore (194d) reported that individual differences
occurred within a group of several breeds of dogs and
because of these diversities in glandular structure,
the genetic make up of the dog played a role in the
histol03y of the thyroid.

The information gathered from the control group
Should add to the literature on the normal histology

of the dog.

REVIEW OF LITERATURE
Thyroid

It was some two thousand years ago that the
thyroid gland was discovered to be a distinct anatomical
entity. According to Hoskins (1941) the Greeks regard-
ed it as a protective device to keep the throat warm
while others thought it was used to round out the neck
so as to make a beautiful contour. Hoskins further
stated that the thyroid was discovered by Vesalius in
1545, but it was not until nearly a century later that
Wharton gave it a name. The term is derived from a
Greek word meaning "Shield-shaped".

It was as late as 1858 before any basic research
was done on the thyroid. At that time, Schiff of
Geneva began research on its function by removing the
gland from experimental animals (Hoskins 1941). Another
twenty years passed before clinicians began to notice
symptoms of thyroid insufficiency in human patients.

Goiters were treated by radical operations after
antiseptic surgery was introduced. The oral administra—
tion of thyroid first began in the latter part of the
nineteenth century.

In 1659 Thomas nharton first accurately described
the deveIOpment of the thyroid gland (Goldsmith 1949).

He stated that the thyroid arises as a downgrowth of

-3...

epithelium from the ventral wall of the pharynx be-
tween the first and second pharyngeal pouches. With

the forward growth of the pharynx, the gland primordiun
appears to grow caudally, dividing into a right and

left lobe. It remains attached to the pharynx by a
stalk or neck, the thyroglossal duct. This thyroglossal
duct connects the young thyroid with the posterior
border of the developing tongue. The thyroglossal duet
atrophies, but the site of origin remains as the foramen
caecum in post uterine life;

According to Gudernatsch (1949), all glands now
"ductless" once had an open secretory duct. The thyroid
at one stage existed as a "duct gland", probably as a
gland auxillary to metabolic function even after losing
direct connection with the digestive tract.

The adult thyroid is composed of small sacs or
follicles. These are considered to be the secretory
units of the gland (leans 1948). Uhlenhuth (1923)
reported that the cells of the follicles produced
the hormone which was stored in the lumen. He consid-
ered the function of the thyroid from two different
aspects; a colloid storage phase and a colloid release
phase.

Bensley (1916) stated that the colloidal secre-
tion was formed in the outer pole of the cell and exb

creted from it directly without passing by the indirect

- 4 -

route through the follicular cavity. The indirect
mode of secretion consisted of the condensation of the
colloid into the form of droplets having a high content
of solids. I

Williams (1944) studied living thyroid cells
and follicles and observed that each cell in a follicle
had three surface regions; one in contact with the col—
loid, one in contact with or close to blood vessels
and connective tissue, and one in contact with adjacent
cells. Williams concluded that when hypertrOphy or
hyperplasia occurred, with loss of typical follicular
structure, the colloid was found between the cells.
As a result of this, a total or partial destruction of
the cells occurred. The connective tissue and blood
vessels grew in from the surface of the follicle until
the necessary conditions of normal contact stated above
were established. The factors which regulated follicular
structure in the gland were as follows: (1) There
was a lethal sensitivity of the thyroid cells to col—
loid in contact with any surfaces other that apical.
(2) The thyroid cells must have their basal surfaces on
or very near blood vessels. (3) The thyroid cells were
supported by a connective tissue investment. (4) There
was polarization of the cells with secretion toward
the lumen. (No evidence was obtained that there was se-

cretion at the base of the cells. Williams concluded

_ 5 _
that colloid is released from the follicle solely by
diffusion across the follicular well.

According to Dvoskin (1948), Verson, using fresh
tissue, was the first to describe the presence of col-
loid droplets within the thyroid epithelium. Recently
a relationship has been established between the form-
ation of intracellular colloid droplets and the thyro-
trOphic hormone.

DeRobertis (l94l) demonstrated intracellular
colloid particles in the thyroids of rats. Using the
freezing-drying method of fixation, DeRobertis expressed
the opinion that the intracellular colloid was distributed
in the following forms: (1) In secretion drOplets at
the apical zone; (2) in large drOplets lying in rows
between the poles of the cell; (5) in pale-staining
base vacuoles; (4) completely loading the cytOplasm,
generally accompanied by pyknosis of the nucleus and
desquamation into the lumen. DeRobertis postulated
that the thyroid cell was able to secrete directly
toward the basal pole, particularly when the gland was
greatly activated in its secretory function. Upon an-
alysing the activation of the cells of the thyroid,
DeRobertis suggested that the thyroid cells produced
an apocrine secretion, because the cytOplasm bulged
into the lumen and was released by the rupture of the

pedicle. After this phase, the cells stOpped secretion

_ 5 -
toward the lumen, and secretion toward the base began,
reabsorbing the colloid stored in the lumen.

Ultracentrifugation has shown that thyroglobulin
has a molecular volume of 675,000 and therefore is a
macromolecule and unable to pass through cell membranes
(DeRobertis 1949).

DeRobertis (1949) studied the problem of the
relation of colloid release to the proteolytic activity
of this material. By extracting proteases by microdis-
section from single follicles, DeRobertis proved that
the follicular colloid was digested by enzymatic action
and the products were absorbed by the cells. It was
also found that thyroid stimulating hormone increased
the proteolytic activity, while iodine administered
for a long period of time decreased it. DeRobertis
(1949) postulated that the release of the secretion
stored in the follicles takes place only after hydrolysis
of the therglobulin inside the follicular cavity.

DeRobertis (1949) reported that the amount and
disposition of the intracellular colloid varied with
species, age, season, function and pathological state.

In the case of hyperactive glands, all of the colloid
particles of a cell were at the base, and the cell

showed a complete inversion of polarity. It was further
stated that this quantity and disposition of intracellular

colloid was a sensitive index of the functional activity

- 7 -
of the gland, and that these changes reflected the
amount of circulating hormone. Therefore, DeRobertis
expressed the Opinion that this could be used as a means
of thyrotrOpic hormone assay.

Dvoskin (1947a) tested different common fixatives
for their ability to preserve the intracellular colloid
droplets. He found that Carnoy's fluid resulted in
the best histolOgical fixation, but found variation
in staining reaction following that fixative. Some
non-staining vacuoles were seen in the stimulated glands,
but whether these droplets represented colloid particles,
as DeRobertis maintained, or whether they represented
artifacts could only be ascertained by a comparison
with the freezing-drying method described by DeRobertis
(1941).

Dvoskin (1947b) experimented with the thyroids
taken from two- to five-day old cockerels. The glands
were incubated in vitro in a variety of media at dif-
ferent temperatures for various periods of time and
under numerous atmospheric conditions. Chemical agents
added to the media included 12.NaI, thyroxine, thiouracil,
thyrotrophic hormones, NaCN, KSCN and KMN04. Dvoskin
found that under most conditions numerous large drap-
lets of intracellular colloid formed in the absence of
added thyrotrophic hormone. This droplet formation

occurred almost entirely in the center of the gland.

- 8 -

The intrafollicular colloid of thyroids treated
with common fixatives and stained with Hallory-Azan
present a variety of colors, including blue, red or
orange (DeRobertis 1941). These variaticns have been
said to be due to iodine content, concentration of
colloid, or fixation methods used. According to
DeRobertis, the freezing-drying method always produced
blue colloid throughout the sections stained with
Xallory-Azan. It was concluded that these variations
found in technics other than freezing-drying, were due
to the method of fixation. With customary fixatives,
clear vacuoles appeared, but in the freezing-drying
method these did not occur. LeBlond (1949) stated
that vacuoles and the blue-red-orange staining reaction
of the colloid were artifacts, but had a definite mean-
ing. Vacuoles were most numerous and colloid was pre-
dominantly baSOphilic in activated or stimulated glands,
while in resting organs the colloid was acidOphilic.
LeBlond postulated that vacuoles and bas0philia in the
colloid corresponded to preperties of colloid usually
found in activated glands.

Nonidez (1932) reported the parafollicular cells
‘to be a second epithelial component of the thyroid gland
of the dog. These parafollicular cells were described
by Nonidez as large epithelial cells with argyrophile

granules. The cells lie in the interstitial spaces in

- 9 -
close proximity to the follicular epithelium from which
they arose. They are called parafollicular cells be-
cause the term does not suggest any functional inter-
pretation. The argerphile granules of the parafollicular
cells differ from those contained in the cytOplasm of
the follicular cells in that they are more numerous and
have a greater affinity for silver nitrate.

It has been established that the thyroid status
can be completely controlled by oral administration of
synthetic materials. Varying degrees of hyperthyroidism
can be produced by feeding iodinated casein, and a hypo-
thyroid condition can be brought about with thiourea,
thiouracil and their derivatives (Blaxter 1949).

According to Cortell (1944), working with guinea
pigs, the effect of exogenous thyroxine upon the thyroid
gland of the intact animal was to put the g and in a
resting state. This was characterized by decrease in
total size, diminution in height of thyroid epithelium
and an accumulation of colloid. I

Astwood (1949) reported evidence for direct effect
of thyroxine in inhibiting the action of thyrotrOphin
upon the thyroid gland. Thyroxine decreased the effect
of injected thyrotrOphin in hypOphysectomized rats. If
extra thyroid hormone was administered, there was a

compensatory atrOphy of the thyroid gland.

..10 -

Anterior Pituitary

The pituitary gland or hypophysis has been known
since the time of Galen (200 A.D.). Vesalius named it
through an erroneous theory of its function. It was
thought that this g and secreted a fluid to lubricate
the throat via minute channels coursing through the
porous cribriform plate of the ethmoid bone. Later it
came to be regarded as a vestige.

Gigantism was first associated with pituitary in-
volvement during the middle of the 19th century, but it
wasn't until 1886, that the French neurologist Pierre Marie
finally determined the relationship of the pituitary to
the disease. Since that time modern medicine has made
great advances in correlating the relationship of the
pituitary with the other endocrine organs and their
respectiVe dysfunctions (Hoshins l94l).

The first suggestion of the pituitary in the
embryo is an outgrowth of Rathke's pouch which gradually
extends toward the brain (Arey l947). As the embryo
grows, this pouch makes contact with a downgrowth from
the brain, the infundibulum. Rathke's pouch ultimately
gives rise to the anterior lobe or pars distalis, While
the infundiblar part gives rise to the posterior lobe
or pars nervosa. The pars intermedia is formed as the
dorsal wall of Rathke's pouch surrounds the infundibulum.

The pars nervosa is intimately coated by the intermedia

_ 11 -
and is easily detached from the pars distalis. The
pars tuberalis does not completely surround the short
infundibular stalk and the upper border of the pars
distalis extends above the mid portion of the pars
nervosa (Plate I). According to Stockard (1941), a
common feature of the English bulldOg is the persistence
of a membranous connection between the anterior lobe
and a depre sion in the basisphenoid bone and in rarer
cases a complete foramen is present pernitting the direct
continuation between the glandular pituitary, Rathke's
pouch and the oral epithelium. The gland lies in a
recess in the sphenoid bone known as the sella turcica.

The anterior lobe consists of solid cords or
nests of cells separated by capillary or sinusoidal blood
vessels. The cells of the anterior hypOphysis are usually
classified in three groups; chromophobes, baSOphils,
and acidophils. The quantitative value for each of
the three types has been worked out for different species.
The values vary with dysfunctions of the pituitary as
well as other endocrine organs.

The morphology of the pituitary of the English
bulldog differs markedly from that of breeds of long
muzzled dogs (Stockard 1941). He reported the pars
nervosa to be thickened, with its dorsal surfaces covered
by a thin layer of the hypOphyseal sac forming a double

layer of the pars intermedia.

-12...

Plate I

INFUNDBULUH
FIRS TUIERALII

INUNI NERVOSA
“KIHIHDI LOID

 

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GLEFT

FIRS Dfiflfllfli
(Alflfllflllfllt)

“II"V

   

MMTIC DRAWING OF W93 CEREBRI

It has been found that the infundibular portions
and the pars intermedia are larger in the human male
than in the female, even though the pituitary as a whole
is larger in the female, because of the enlarged pars
distalis. Stockard (1941) found the pituitary of the
English bulldog to have a small pars distalis and a
very large pars intermedia and pars nervosa. On the
basis of the human findings he classified the pituitary
of the English bulldog as strongly of the male type.
Stockard (1941), postulated that the masculine prepor-
tions of the bulldog pituitary may account for the fact
that the female bulldog produces only small litters
and displays defective maternal instincts. He further
warned, however, that there was also frequent histo-
pathology in the tuberalis, as well as low basephil
counts, which might account for such masculine tendencies.

//Stockard (1941) reported that the English bulldog
pituitaries contained an abnormally small amount of
secretory epithelium in the pars tuberalis and pars
distalis. The acidophils were bright-staining and in
the high preportion of thirty acidOphils to one basophil.
Stockard (1941) described the baSOphils as being frequently
vacuolated, and in the form of the "signet ring" type
of castrate cells.

”In a study of the cyclic histological changes
in the anterior pituitary of the female dog, Wolfe 33 31.

(1983) described four cell types and designated them

- 14 -
with Roman numerals. Type I cells were the acidOphils
or alpha cells, type II and type III were both of the
bas0philic type, but exhibited certain separate charact-
eristics and the last group, type IV, were the chromo-
phobes. The cells of type I were highest during anestrus,
type II and III reached their peak at ‘estrus and type
IV were most numerous during the late lutein phase.

In differentiating the four cell types accord-
ing to the erythrosin-Orange G-aniline blue method of
Cleveland and Wolfe (1932), Wolfe 33 El. (1933) reported
that the granules of type I cells stained sharply with
Orange-G and the nucleus was light blue; the granules
of type II cells stained red-orange. Type III cells
were divided into the granular and non-granular. The
granular cells appeared as dark blue cells filled with
diffuse granules usually on the periphery of the cytOplasm.
These cells were classified as type III because of these
small clumps of granules and also due to the intact
cell membrane and cytOplasm. Type IV cells were those
which corresponded to the chromOphobes. These cells
arose as a result of the loss of granules and subsequent
regression of the cells with granules. dolfe 23 El.
(1938) stated that all granular cells pass into cells
of this type.

In 1938 Dawson and Friedgood discovered a fourth

cell type in the anterior pituitary of female rablits

.‘." 15 _
and cats with special affinity for azocarmine. These
findings were followed by investigators finding similar
cells in the anterior lobes of other Species. They
have been reported in the anterior hypophysis of the
Opossum, ferret, and monkey by Dawson and in the
armadillo by Oldham (Eartmann t al. 1948).

.- ———-.

fiartmann it fil' (1946) confirmed the eristence of

a fourth cell type in the do: as described by Volfe et F1.

in 1933. fiartnann et 81. (1946) studied the relation

inev concludvd th;t this fourth cell tfpe, which they
calllr7 the olgfiixrtsin celd_llecause tuna “rinldrwt:9tained
selectively with erytorhsin, cannot be vioved as a mere
variant of another chronopnilic tVTS.

Grinch cl amid Plrves (1945) unfiflrfiu ts rs their
erperimenthl Shingle, induced extreme thyroxine deficiency
and found complete denranulation of acidophils with an
increase in baSOphils. When a low dose of thyroyine

was administered (3.0 ug/lQUg/day), baSOphil numbers
decreased, revealinr evidence of activation. When this
dose Was increased to 3.35 uv/loUg/day the basophils
were reducel in size and the granules stained purple.
They concluded that the thyrovine requirement of a rat
was eouivalent to b.25 ug/lUUg/dny. The thyroxine re-

quirement as it effects the pituitary function is the

amount of tovroxine equivalent to the normal daily

- 16 -
thyroid secretion in an animal receiving adequate amounts
of iodine. Any slight reduction in normal thyroxine
level causes an increase in numbers and cytological
activity of the basophil cells of the pituitary, (Gries-
baeh and Purves 1945). These same authors concluded
that this activation of the baSOphilic cells is corre-
lated with increased secretion of thyrotrOpin and there-
fore these baSOphil cells are a source of thyrotropic

secretion.

Adrenal

The existence of the adrenal glands was first
known to science in 1563 when they were described by
Eustachius. It wasn't until 1855 that the functionsof
the adrenal glands were first described by Thomas Addison.
This was followed by animal experiments conducted by
Brown-Sequard in which he removed the adrenals from dif-
ferent animals and recorded the subsequent effects
(Hoskins 1941).

In 1895 two British workers prepared extracts
for use in treating patients and observed the effect
upon the tone of the heart and arteries. This was the
beginning of the work which led to the production of
epinephrine or adrenalin.

According to Hoskins it was in 1905 that Elliot

made some basic observation on the effects of the ex-

- 17 _
tract of adrenal medulla which later proved to be the
basis for Cannon's "emergency theory".

The adrenal consists of two parts: a central
portion, the medulla, and a surrounding outer portion,
the cortex. According to Hoskins (1941), the two parts
are of different embryological origin.

The cortex is derived from early mesoderm in the
anterior part of the embryonic body cavity. The inter-
renal buds are the first suggestion of the future cortex.
These cells coalesce into a larger structure.

The medulla arises from groups of cells of the
abdominal plexus of ectodermal origin. These cells,
called pheochromoblasts, invade the primitive interrenal
structure, and take a position in the central part of
the organ (Hoskins 1941).

The thyroid-pituitary-adrenal relationship is
shown on plate II. It is clearly pictured that, in
general, thyroxine stimulates the adrenal cortex.

Preston (1928), using mice, studied the effect
of thyroxine on the adrenals. She reported the size
of the cortex to be twice the normal size with the retic-
ular zone showing greatest hypertrOphy. {arked hyperemia
was also reported by this author.

Herring (1917) administered small quantities of
ox thyroid to female white rats and found an increase

in the size and weight of the suprarenals. He stated

- 18 -
that both the cortex and the medulla were enlarged but
that the hypertrOphy in the cortex was greater than in
the medulla.

Deanesly (1951) submitted several mice to varying
doses of thyroxine. She reported all adrenals to be in-
creased in size fifty per cent with the cortex showing
most of the enlargement. It was found that the fat
disappeared first from the fascicular zone and had a
tendency to accumulate around the medulla.

In 1938 Schmidt and Schmidt studied the variation
in the structure of the adrenals produced by thyroxinc
and at various environmental temperatures. They found a
large increase in the number of mitotic figures with
the greatest amount in the fascicular zone in the animals
kept at 200°

In an effort to find a method for estimating
the increase in the adrenocortical secretion rate in
hyperthyroid rats, Wallach and Reineke (1949) attempted
to prevent the response of the adrenals to thyroxine
by the simultaneous administration of adrenocortical
hormone. The dosage necessary to accomplish this
would be some indication of the increase in the secre-
tion rate of the adrenals. They regarded this hyper-
secretion of the adrenals as strong evidence for an
increased requirement for adrenocortical hormone. Be-

cause the adrenals were enlarged after thyroxine ad-

~- l9 -
ministration, more adrenocorticotrophic hormone was
secreted (Tallach and Reineke l949).

Hartman and Brownell (1949) reported that any
increase in the thyroid hormone produced adrenal hyper-
trophy. They stated that there was cell increase in
both number and size in all zones. The reticular zone
sometimes doubled, and if removed regenerated. These
same authors found over three times as many mitoses in
thyroxin-treated mice. These mitotic figures occurred
mostly in the fascicular zone.

The evidence of the precise relationship of the
thyroid to either part of the adrenal is not complete.
Hartman and Brownoll (l949) stated that the thyroid
was essential for the full effect of adrenooorticotrophic
hormone since the influence the pituitary exerts on
the adrenal was greatly diminished if the thyroid had
been removed. (Plate III) Means (1948) claimed that
there were indications of some sort of antagonism be-
tween the hormone of the adrenal cortex and the thyroid.
Therm, according to Heans (1948), reported that thyrotoric

rats ai en adrenal-cortical extract had less body weight

V

4

loss and less increase in cardiac weight.

Hartman and brownell (1949) stated that thyrorine
acted through the pituitary since it was not effective
after hypophysectomy. This rhenomenon is not entirely
understood or completely explained and has left many

questions unansrered.

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Plate III

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ANT. PlTllTARY
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THYROID ACTS AS A CATALYST IN ACTH mono»

KATERIALS AED KETHCDS

The initial work of Bergman and Reineke (1949)
was done in the fiichigan State College Department of
PhysiOIOgy, as a part of the work for a master's degree.
A portion of the experimental detail is reviewed here
as background material.

Seven English bulldog puppies were used in this
experiment. There were four degs in the control group:
1, 5, 5 and 6. The protamone group consisted of three
dogs: 2, 4 and 7. Dogs 1, 2, 3 and 4 were males and
5, 6 and 7 were females. Animals 1 and 2 were sixty-
four-day-old littermates, weaned fifteen days prior to
the beginning of the experiment. Degs Z, 4, 5, 6 and 7
were fifty-three-day old littermates, weaned two days
previous to the initiation of the trial. Deg 3, placed
on the same bitch as l and 2, received more nourishment
than his littermates and grew faster. All animals were
in good condition at the beginning of the experiment.
Their ancestry indicated that they were a small type
English bulldog.

The diet consisted of condensed milk, canned dog
food, *"Pard" and dry meal, **"Gr0pup". The exogenous
thyroid used for this investigation was protamone,
the commercial name for the product supplied by Cero-

phyl laboratories. It is a synthetically iodinated

 

* . q . . .
**"Pard" - Swift and Company, Chicago, IllihOis
"GrOpup" - Kellogg Company, Battle Creek, Hichigan

- 23 _
casein. For the dogs in the experimental group, 4 gm.
of Protamone were mixed to every 100 lbs. of dry feed.
The three experimental dogs consumed a total of 30.269
lbs. of dry matter and 21.345 gm. of protamone. The
trial lasted approximately 5 months.

On the 48th day of the trial, radioactivity
studies were done. They indicated a lessened pickup
of iodine by the thyroid glands of the protamone group.
This was explained by the suppression of thyroid function
by the exogenous thyroid.

For further details of the feeding experiments,
the original paper by Borgman and Reineke (1949) may
be consulted.

After the experimental period ended, the dogs
were kept on a normal diet for approximately a month.

They were sent to the Department of Anatomy for autOpsy
in July at the age of eight months. The dogs were an-
esthetized with sodium pentathol and bled out from the
internal carotid artery. The thyroids, anterior pituitary
and adrenals were secured from each dog.

The tissues were fixed in Bouin's fluid and de-
hydrated and infiltrated according to the butyl alcohol-
paraffin mush method, Johnson 23 2l°’(1943)°

The blocks were sectioned at 7'u and representative
slides for each organ stained with hematoxylin-eosin.
Other stains were used on certain sections, the stain de-
pending on the structure under study. These stains will be

discussed under the individual organs.

RESULTS AND DISCUSSION

Thyroid

Plate IV depicts the effect of protamone on the
cytology of the thyroid. There is a general accumula-
tion of colloid and decrease in the epithelial height.

The thyroid-stimulating hormone which is produced by

the anterior pituitary is inhibited by the protamone

and subsequently the output of the thyroid hormone is
decreased.

Each section of thyroid was stained by the Mallory-
Azan technic as described by DeRobertis (1941). The
epithelial height was measured by means of an ocular
micrometer. Twenty-five follicles in each of the seven
thyroid sections were measured and the average values
computed. The mean epithelial height in follicles of
the three experimental dogs was 6.50 u. The mean epithelial
height of the four control animals was 11.90 I. This con-
stituted a 54.53% decrease in cell height as a result of
the protamone given. (Plate V)

The data were submitted to a statistical analysis
of variance. The analysis showed a very highly significant
variance due to the treatment. The dosage of protamone
produced prominent histological changes in the thyroid
epithelium.

There was a significant difference between the
animals, indicating a variation in epithelial height be-

tween the individual dogs irrespective of dosage. However,

_ 25 -
the difference in epithelial height between the control
and the experimental groups was so large as to overshadow
the differences between animals. (Plate V)

The accumulation of colloid in the follicles is
usually associated with the response of thvroid to thyroxine.
All the follicles were well filled with colloid; however,
no marked accumulation was noted. (Plates VII and VIII)

Vaeuoles were present in thyroi<h from both groups
but were more numerous in the control group. Intracellular
colloid was not identified for certain in any of the
sections.

Parafollicular cells common in the thyroids of
most dogs were not seen. This supports the Opinion of
Stockard (1941), that these cells are practically absent
from English bulldog thyroids.

The physiology of the English bulldog disputes
the idea of thyroid hyperactivity. The basal metabolic
rate is low and the animal is usually inclined to be
slow, inactive and fat, although a comparatively small
eater (Stockard 1941).

Stockard (1941) reported that the thyroid of the
English bulldog contained an excessive amount of extra-
follicular material consisting of epithelial cells not
preperly incorporated into the follicles. The author
did not find this to be true. The follicles were well

formed with distinct connective tissue bands separating

- 26 _

them. This difference might have been due to seasonal
or climatic differences. These animals were destroyed
during summer months; however,the temperature was not
exceedingly high. It may be of some interest to mention
that they were housed in a brick Luilding and not sub-
mitted to the direct sun or allowed to run in Open kennels.

The degree of thyroid suppression obtained in this
experiment was somewhat limited. Radioactive iodine de-
terminations revealed only a 24% reduction on the function
of the thyroid of those on protamone. This indicated
that the thyroid was not suppressed to such an extent
that it would not be able to resume normal function when

thyroid administration was stOpped.

-27..

Plate Iv

  

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EFFECT OF THYROXINE ON THE CYTOLOGY OF THYRGD

-28-

Plate V

MEASUREMENT OF FOLLICULAR EPITHELIUM
Figures Computed on 25 Measurements

ANALYSIS of VARIANCE

 

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Animal No. l 3 5 6 2 4 7
Sex d 9 9 Q

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Animals

Plate VI Thyroid. Mallory-Azan stain. 165K.

Fig. 1. Experimental animal. Female.
Fig. 2. Control animal. Female.

. 29 -

Plate VI

 

Fig. 1

 

Plate VII

Thyroid. Mallory-Azan stain. 530x.

Fig. 1. Experimental animal. Female.
Fig. 2. Control animal. Female.

These views illustrate the effect
the protamone produced on the cytology
of the thyroid. The control has many
vacuoles in the colloid and taller
follicular epithelium. The experiment-
al animal shows a general accumulation
of colloid, lowered follicular epithelium
and fewer vacuoles.

30-

Platc VII

 

Fige 1

 

Fig. 2

Plate VIII

Thyroid. Mallory-Azan stain. lOOOX

Fig. 1. Experimental animal. Female.
Fig. 2. Control animal. Female

These are kodachrome prints of
the same area as that shown on Plate VII.

-31-
Plate VIII

 

Fig. 2.

-52..

Anterior Pituitary

The relationship between the thyroid and the
pituitary and the effect thyroxine produces is shown in
plate IX. When excess thyroxine is administered the
numbers of bas0phils are reduced.

In an effort to determine the effect of protamone
upon the English bulldOg pituitary, several different
stains were employed in addition to the routine hema-
toxylin-eosin stain. MacCallums's modification of
Cowdry's cogper-hematoxylin stain for the pituitary gland,
Mallory (1942), was used to differentiate the cells of
the pars intermedia from the basophilic cells of the
anterior lobe of the pituitary. (Plate X) The fourth
cell type described by Wolfe gt El. (1933) was brought
out by the Erythrosin-Orange G-aniline blue method of
Cleveland and Wolfe (1952). (Plate XI) An attempt was
made to emphasize the bas0phils and acidOphils with
Masson's trichrome stain, Mallory (1942).

There were very few bas0phils present in the sec-
tions of the controls. The slides stained with the cepper-
hematoxylin stain revealed some cells considered to be
of the bas0philic type. However, counts made in numerous
areas throughout the sections gave inconsistent results.
The experimental sections were almost completely depleted
of bas0phils. Because of this, any attempt to differenti-

ate control from experimental animals by means of the

 

as

“ou-

differential count was abandoned. This nearly complete
absence of basophils in the pituitary of the English
bulldog supports Stockard's (1941) findings of excessive
acidophils and few basOphils.

The sections stained with Masson's stain showed
a large accumulation of acidOphils in the experimental
group (Plate XII, fig. 2) when compared to the control
(Plate X11, fig. 1). These Masson-stained sections were
compared with a section of canine anterior hypophysis
(breed unknown) prepared at the University of Minnesota,
Department of Veterinary Anatomy. (Plate XIII) Undoubt-
edly this section was not from an English bulldog since
four cell types were very prominent. The bas0phils were
sharp and distinct from the bright red stained acidophils.
The chromophobes were colorless and another cell with a
pale staining cytoplasm was presumed to be the fourth
cell type reported for the dog.

It is reasonable to assume that the protamone re-
duced the basophil cells since the treated group contained
larger numbers of acidophils when compared to the sections
of the untreated group.

The histological sections showed some evidence
that the fourth cell type, described by several workers,
might exist in the English-bulldog pituitary. Different
fixation methods than those employed here, should aid in

clarifying this point.

~— 54 -

Low beSOphil counts in the English bulldog indicate
thyroid hypofunction and may account for the small litters
produced by these dogs. Stockcrd (l941) classified the
pituitaries of English bulldog as being of the male type,
because of this abnorxal acidophil-basophil ratio. Diets
supplemented with protamone in growing English bulldogs
and other brachycephalic breeds could possibly result

in the production of larger litters.

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Plate IX

 

 

 

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Plate X

Anterior Pituitary. COpper-hematoxylin stain.

Fig. 1. Control animal. Male.
Fig. 2. Experimental animal. Hale.

These views illustrate the presence of a
few baSOphils in the anterior pituitary of the
control compared to the complete absence of
bas0phils in the anterior pituitary of the ex-

perimental animal. The large, spheroid, black-

stained cells in the center of Fig. l are
baSOphils.

560K.

 

Plate XI

Anterior Pituitary. Erythrosin-orange-C-aniline

blue stain.

This is a
terior pituitary
illustrates this
printing process
has been lost.

760K

kodachrome print of the an-
from a control animal which
Special stain. Due to the
much of the color detail

-37-

Plate XI

 

Plate XII

Anterior Pituitary. Kasson's trichrome
stain. 7eox.

. 1. Control animal. Male.
. 2. EXperimental animal. Male.

talk.)

09‘ CH

1
1

These kodachrome prints illustrate
the decrease in basophils produced by
the protamone. Fig. 1. shows one baso-
phil (a) and numerous chromophobes,
while fig. 3. shows no basophils and
numerous acidOphils. The printing
process has reduced the color differ-
entiation.

l

-158-

Plate XII

 

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468-

Plate XII

 

Plate XIII

Anterior Pituitary. masson's trichrome stain.
7oJX.

This is a kodachrome print of a slide
prepared in the Veterinary Anatomy Depart-
ment of the University of Kinnesota. The
breed and sex of this dog was unknown.

A. hasophil.

B. Acidoohil.

C. ChromOphobe.

D. Fourth cell type described by
Cleveland and Wolfe.

-39-

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Plate XIII

 

- 40 -_

Adrenal

The adrenal glands were stained with Kasson's
triple stain. The were sectioned longitudinally and
measurements were made in the areas shown on plate XIV.
The entire cortex of each gland was measured as well as
the component zones.

Analysis of variance of the measurements on the
cortex indicated a highly significant difference due to
the treatment. However, no difference was found between
the individual animals within each group. When the
analysis was applied to the individual zones, the same
results were obtained. However, it should be noted that
the glomerular zone was smaller in the treated animals
than in the controls while the reticular and fascicular
zones increased in size with the treatment. (Plate XV)
This cortical hypertrophy produced as a result of the prO-
tamone, was probably due to hypersecretion of the adrenal.
It has been said that the thyroid is essential for the
full effect of adrenocorticotrophic hormone from the
pituitary. It could be said that the thyroid acts as
a sort of catalyst in this adrenal-pituitary relationship.

The thyroid and adrenal medulla appear to be mutual-
ly stimulating. Increased activity of one stimulates
the other. Karked hyperemia of the medulla was seen in
all adrenals of the experimental animals, while no hyper-

emia was present in any of the adrenals from the control

_ 41 _
animals. (Plate XVI) This same result was reported by
Preston (1928). The exact mechanism of this hyperemia

is not entirely understood.

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Plate XV

MEASUREMENTS OF THE ADRENAL cosTEx
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ANALYSIS of VARIANCE
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Plate XV

Adrenal Cortex. Masson's trichrome stain. Male.
95X.

These illustrate the cortical hypertrophy
produced by the protamone. Compare the width
of the cortex in Fig. l with that in Fig. 2.

Fig. 1. Control animal. Male.
A. Zona Glomerulosa. '
B. Zona Fasciculata.
C. Zona Reticularis.
D. Beginning of Medullary portion.

Fig. 2. Experimental animal. Male.
A. Zona Glomerulosa.
B. Zona Fasciculata.
C. Zona Reticularis. The entire reticularis
is not shown.

..44...

Pl ate XVI

 

 

 

 

 

 

-44-..

Pl ate XVI

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Plate XVI

Adrenal Xedulla. hasson's tricnrome stain.
l90X.

his. 1. Control animal. Yale.
Fig. 3. EXperimental animal. Sale.

These kodachrome prints illustrate
the hyoeremia present in the adrenal
medulla of the experinental animal. There
is no blood in the medullary sinuses of
the control group.

-44a-

Plate XVII

 

 

2.

Fig.

S Till-HY All) CLIICLUSIOITS

Seven purebred English bulldog puppies were used
in this experiment. They were divided into two proups,
separated according to sex and littermates. Three were
experimental dogs and were fed a total of 21.345 gms
of protamone over a five month period. Four were the
control animals anl they were fed exactly as the others,
omitting the protamone.

. They were autOpsied at eight months of age and
histOIOgical studies were done on the thyroid, anterior
pituitary and the adrenal.

The thyroids of the experimental group revealed
a generalemcumulation of colloid and a 54.53% decreas
in follicular epithelial height, when compared to those
of the control group. Statistical analysis of the data
showed a highly significant variance due to the protamone.

The baSOphils in the anterior pituitary were de-
creased at the expense of an increase in acidOphils.
Formal anterior pituitaries of English bulldogs contained
very low numbers of baSOphils. The anterior pituitaries
of the experimental dOgs were almost completely depleted
of baSOphils. Because of this breed difference, differ-
ential counts were attempted, but finally abandoned.

The adrenal glands of the experimental animals
showed a marked cortical hypertrOphy. Keasurements of

the cortex were made on all adrenals. The data were

- 4e -
analyzed statistically and proved to show a highly sig-
nificant variance. Hemorrhage into the sinus spaces of
the medulla was present in the adrenals of the experi-
mental animals, but not present in the control dogs.

It is obvious that a low dose of protamone such
as this, given to a normally hypothyroid animal will
produce significant histological changes in these par-
ticular endocrine organs.

The thyroid activity of the English bulldog is
low and histologically shows a picture of general hypo-
function. After protamone administration this activity
was further diminished and the gland was put in a resting
state.

The anterior pituitary of the English bulldog
contains very low numbers of baSOphils. After exagenous
thyroid was given, these basophils decreased in numbers
still further and the gland was nearly depleted of them.
This absence of basophils was due to the presence of
protamone in the body indicating that the basophil cell
is a source of thyrotropic secretion.

Exogenous thyroid stimulated the cortex of the
adrenal cauSing hypertrophy, with the reticular zone
showing the greatest increase. This effect was apparent-
ly mediated through the anterior pituitary, since it
must be present for the full effect.

Protamone could possibly be used as a supplement

to the normal diet for dogs of those breeds with tendencies

toward a hypothyroidism. This treatment might result

in increased litter size.

LITERATURE CITED

Arey, L. B., 1947. Developmental Anatomy. W. B. Saunders
Company, Philadelphia.

Astwood, E. B., 1949. Mechanisms of action of various
antithyroid compounds. Annals of New York Acad.
of Sciences, 50:419—443.

Bensley, R. R., 1916. The normal mode of secretion of
the thyroid gland. Am. J. Anat., 19:37-42.

Blaxter, K. L., E. P. Reineke, E. W. Crampton and W. E.
Peterson, 1949. The role of thyroidal materials
and of synthetic goitrogens in animal production
and an appraisal of their practical use. J.

An. Science, 8:506-352.

Borgman, R. J. and E. P. Reineke, 1949. The response
of English bulldog puppies to thyroidal stimulation.
J. Am. Vet. Med. Assoc., 115:480-486.

Cleveland, R. and J. M. Wolf, 1932. A differential stain
for the anterior lobe of the hyp0physis. Anat.
Rec., 51:409-413.

Cortell, R. and R. W. Rawson, 1944. The effect of thyroxine
on response of the thyroid gland to thyrotrophie
hormone. Endocrinology, 35:488-498.

Dawson, A. B. and H. B. Friedgood, 1938. Differentiation
of two classes of acidophils in the anterior
pituitary of the female rabbit and cat. Stain
Tech., 13:17-21.

Deanesly, R. 1931. The histology of adrenal enlargement
under experimental conditions. Am. J. Anat.,
47:475-498.

Dempsey, Edward W. 1949. The chemical cytology of the
thyroid gland. Annals of New York Acad. Sciences,
50:536-357.

DeRobertis, E., 1941. The intracellular colloid of the
normal and activated thyroid of the rat studied
by the freezing-drying method. Am. J. Anat.,
68:317-337.

DeRobertis, E., 1949. Cytological and cytochemical basis
of thyroid function. Annals of New York Acad. of
Sciences, 50:317-335.

- 49 -

Dvoskin, Samuel, 1947a. Intracellular colloid droplets
as a basis for thyrotrOpic hormone assay in the
chick. Endocrinology, 41:220-229.

Dvoskin, Samuel, 1947b. The spontaneous formation of
intracellular colloid droplets in surviving chick
thyroid tissue. Endocrinology, 41:403-416.

Dvoskin, Samuel, 1948. The effects of pituitary and
non-pituitary gland factors on the formation of
intracellular colloid droplets in the thyroid
epithelium of hypOphysectomized rats. Endocrin-
ology, 43:52-70.

Gilmore, James W., 1940. Growth changes in body organs.
Part II Growth changes in the thyroid of the
normal dog. Am. J. Vet. Res., 1:66-72.

Goldsmith, E. D., 1949. Phylogeny of the thyroid:
descriptive and experimental. Annals of New York
Acad. Sciences, 50:283-312.

Griesbach, W. E. and H. D. Purves, 1945. The significance
of the baSOphil changes in the pituitary accompany-
ing various forms of thyroxine deficiency. Brit.
J. EXP. Paths, 26:13-17.

Gudernatsch, Fred, 1949. See Discussion of Paper by E. D.
Goldsmith, Phylogeny of the thyroid: descriptive
and experimental. Annals of New York Acad. Sciences,

Hartmann, Frank and K. A. Brownell, 1949. The Adrenal
Gland. Lea and Febiger, Philadelphia.

Hartmann, J. F., W. R. Fain and J. M. Wolf, 1946. A cyto-
logical study of the anterior hypOphysis of the
dog with particular reference to the presence of
a fourth cell type. Anat. Rec., 95:11-27.

Herring, P. T., 1917. The effect of thyroid-feeding on
the weight of the suprarenals and on their adrenalin
content. Quart. J. Exp. Physiol., 11:47—57.

Hoskins, R. G., 1941. Endocrinology: the Glands and their
Function, W. W. Norton and Company Inc., New York,
New York.

Johnson, Eunice, F. N. Andrews, and C. L. Shrewsbury, 1943.
The preparation of muscular tissues for histological
study. J. Animal Science, 2:244-250.

- 5o -

Leblond, C. P., 1949. See Discussion of Paper by De-
Robertis, Cytological and cytochemical basis of
thyroid function. Annals of New York Acad.
Sciences, 50:517-355.

Mallory, F. B., 1942. PatholOgical Technique. E. B.
Saunders Co., Philadelphia.

Means, J. H., 1948. Thyroid and Its Diseases. J. B.
Lippincott 00., Philadelphia.

Nonidez, J. F., 1952. Origin of the parafollicular cell,
a second epithelial component of the thyroid gland
of the dog. Am. J. Anat., 49:479-505.

Preston, M. 1., 1928. Effect of thyroxine injections on
the suprarenal glands of the mouse. Endocrinology,
12:325-534.

Schmidt, I. G. and L. H. Schmidt, 1958. Variations in
the structure of adrenals and thyroids produced
by thyroxine and high environmental temperature.
Endocrinology, 25:559-565.

Stockard, C. R., 1941. The Genetic and Endocrine Basis
for Differences in Form and Behavior. Wistar
Inst. of Anat. and Biol., Philadelphia.

Uhlenhuth, E., 1925. The elaboration and release of the
colloid of the thyroid. Proc. Soc. Exp. B101.
and Med., 20:494-505.

Wallach, D. P. and E. P. Reineke, 1949. The effect of
varying levels of thyroidal stimulation of the
ascorbic acid content of the adrenal cortex.
EndocrinolOgy, 45:75-81.

Williams, R. G., 1944. Some preperties of living thyroid
cells and follicles. Am. J. Anat. 75:95-117.

Wolfe, J. M. and Rucker Cleveland, 1952. Cell types’
found in the anterior hypophysis of the dog.
Anat. Rec. 52:45-44.

Wolfe, J. M., R. Cleveland, and M. Campbell, 1983. Cyclic
histological variation in the anterior hypophysis
of the dog. Zeit. Zellforsch., 17:420-452.