THE EFFECT OF SIMULTANEOUS ADMINISTRATION OF TESTOSTERONE
PROPIONATE AND THYROPROTEIN ON GROWTH AND ON THE MECHANISM
OF PROTEIN METABOLISM IN GROWING MICE

By
Alfred Novak

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
DOCTOR OF PHILOSOPHY

Department of Physiology and Pharmacology

1950

ACKNOWLEDGEMENTS

The author wishes to express his appreciation to the following
members of the Department of Physiology and Pharmacology:

Dr, J,

Meites, Dr, L, 7. Wolterink, and Dr. B, 7, Alfredson for advice and
materials.

Dr. W, D. Baten, Mathematics Professor of the Experiment

Station helped considerably with the statistical analysis.

Mr. J.

Monroe of the Animal Laboratory of the Department of Physiology and
Pharmacology provided valuable service in the handling of animals and
maintenance of a uniform laboratory environment.

Thanks are extended

to Ciba Pharmaceutical Co., Summit, New Jersey for providing testos­
terone propionate in pellet, powder, and oil form,

Cerophyll Labora­

tories, Kansas City, Missouri, provided ample supplies of Protamone.
Particular thanks go to Dr, 7. L. Wynd, Department of Botany, for
valuable technical information and use of his laboratory and equip­
ment for the chemical analyses made of the animals.
Greatest appreciation is due Dr, E, P, Reineke who provided stimu­
lation and advice throughout this work, and above all, gave the author
the utmost freedom in the development of ideas, the attainment of facts
and techniques, and the chance to make mistakes - all of which are es­
sential factors in the development of the independent worker and of
progress in Science.

TABLE OF CONTENTS

INTRODUCTION.................................................... 1
HISTORICAL APPROACH TO THE PROBLEM............................... 3
Growth Effect3 of Male Hormone ,. .

.........................3

Growth Effects of Thyroid Hormone.......................... 13
Effects of Combinations of Male and Thyroid Hormones . . . . .

17

EXPERIMENTAL PROCEDURE AND RESULTS...............................20
Preliminary Investigations ................................

20

General Procedure .....................................

22

.

Experiment O n e .............................................24
Technique.............................................24
Growth D a t a ........................................... 25
Food and water consumption d a t a ........................ 28
Accessory sex organ assay d a t a .........

34

Experiment T w o ............................................. 35
Technique...........

35

Growth d a t a ........................................... 36
Food and water consumption data

.................. 39

Accessory sex organ assay d a t a ......................... 44
Experiment Three

......................

44

Technique.............................................44
Growth data .'.................................

50

Food and water consumption d a t a .............

55

Experiment F o u r ............................................57
Technique................

57

Growth d a t a ........................................... 60

TABLE OF CONTENTS (Cont. )

Accessory sex organ assayd a t a .......................... 63
Food and water consumptiond a t a ......................... 63
Analysis of carcass d a t a ...............................67
DISCUSSION..................................................... 97
SUMMARY AND CONCLUSIONS..............................
LITERATURE C I T E D ...............................................109
APPENDIX.................................

107

INTRODUCTION

The field of growth and organlsmic development has challenged the
curiosity of man for many centuries.

Historically, one aspect of growth,

then another was developed and took precedence so that at one time the
general nutritional factors in growth were the center of interest and
effort, then came the era of calories and energy factors, followed by
the era of vitamins.

At a later date the trace elements and endocrine

glands came to the forefront.

Today, the relationships of the various

glands to one another is receiving considerable attention along with
the basic food substrate requirements of micro-organisms and of other
organisms of comparatively simple nature.

The latter field of study

now occupies the center of the stage of the drama, of growth both from
the standpoint of maintenance and development of the individual and the
prevention of abnormal growth.

Basic research is going on in the area

of biochemistry of growth where the underlying nutritional problems are
not those of general protein, carbohydrate, and fat or water metabolism
but of particular amino acid, glucose-6-phosphate, glyceric acid, vita­
min B requirements, of enzyme activity, of gluconeogenesis, high phos­
phate bond energy transfer, carbon pool turnovers, etc.

In addition,

in the field of endocrinology, one sees a determined search for the play
of forces of the various endocrine glands upon one another, upon the
various tissues, organs, and systems of the body, and upon the various
activities mentioned above.

Among the endocrine glands that are known

to play a role in growth are the anterior pituitary via its growth hor­
mone, the pancreas via its hormone control of glycogen storage and glu­
cose metabolism, the adrenals through growth-stimulating and glycogenstoring action of cortical hormones, and the thyroid and testes via

their hormones.

It is evident from even cursory perusal of the litera­

ture that the interplay of scores of factors must he analyzed and inter­
related if the growth process is to he fully understood.
This paper represents work carried out to determine the effects
of various dosages of thyroidal substances and male hormones when ad­
ministered simultaneously in immature male albino mice.

The stimulus

for this study came from the observations and experiments of the many
workers concerned with the growth problem which indicated that the
separate growth effects of the male hormone and thyroid hormone might
produce synergistic effects when administered together.

HISTORICAL AFERQACH TO THE PROBLEM

Growth Effects of Male Hormone
It is pertinent at this point to discuss the literature related to
the problem.

At the time of writing, no one has attempted direct

studies on the simultaneous administration of testis hormone and of
thyroidal substance but the field of endocrinology shows much basic
work done by investigators interested solely in the effects on growth
of thyroid or testis hormones.
Since ancient times differences in body weight and muscular de­
velopment have been observed between the human male and female.

This

same relationship was noticed in many other mammals and even among
birds so that without much understanding as to the basic principles
involved, castration was practiced and the market value of pigs and
chickens was raised considerably by this procedure.
Other historical evidence of the male hormone deprivation effects,
although again understood only superficially, were observed in the
classical eunuch.

Here the body configuration of a young male eunuch

was little changed from the boyhood softness and muscle distribution
whereas castration of older males resulted in a regression from firm
bodies to flabby, fleshy bodies with increased girdle fat, diminished
genitalia and disproportionately long arms and legs.
A hazy concept of the anabolic effects of the testis may have been
in the mind of Brown-Sequard who in 1889 at the age of 72 injected tes­
tis extracts into his own body and reported a general somatic invigoration as well as sexual stimulation.

Here was a man of profound vision

and courage notwithstanding the subjective nature of his Inquiries.

How­

ever, it wasn't until 1935 that a definite protein anabolic effect of

3

4
androgens was established by Kochakian and Murlin in rats.

Soon there­

after, Kenyon and his group (1938) shoved that the same effect could
be produced in man.

Viewed from another direction Richter (1933) noted

that the removal of the testes produced a decrease of about four-fifths
in the daily running activity of rats.
In 1922 Dr. Carl Moore at the University of Chicago noticed a de­
crease in the growth rate of castrate guinea pigs.

Both the body

weight and bone lengths were smaller in castrates than in normal males.
Moore quotes Steinaeh as saying that "inasmuch as the male of both rats
and guinea pigs is usually heavier than the female, the testis is re­
sponsible for an internal secretion that is liberated by the inter­
stitial cells that promotes growth".

This is the first evidence of

clear-cut thinking concerning the question.

Somewhat later Kbrenchevsky

(1925) reported that in most cases, removal or hypofunction of the sex­
ual glands in animals as well as human beings is followed by an Increase
in weight and an accumulation of fat.
trates (eunuchs).

He called these the "fat" cas­

In approximately 40# of the eases, castration or

hypofunction of the gonads did not produce the above effect.
called the "thin" castrates (eunuchs).

These he

This apparent discrepancy in

results of castration was conducive to further study.

In 1932, Commlns

noted a retarding effect on weight accumulation after castration of
male albino rats.

In 1936, Lawless showed that castrate rats were

shorter and weighed less than the controls.
the ideas of Steinaeh and the work of Moore,

These results verified
Lawless also had a check

on the thyroid weights of his animals in both groups and noted no defi­
nite differences although earlier workers had indicated contrary results.
In the same year, Holt, et al, after a short period of study concluded

5

that the weights of male albino rats following castration varied and
that these variations were of no significance and not predictable.

He

also found no significant difference in food consumption in castrates
as compared to normals.
It was not clearly recognized at the time that a very high dose
of a compound might have opposite effects to those of a small dose,
Rubinstein, et al. (1939b) showed significant depression of body weight
and length of male albino rats by injection of daily 1 mg. doses of
testosterone propionate.

Deansley and Parkes (1941) found growth in­

hibition with 20 mg. pellets of testosterone propionate in rats.
a

McEuen,

et al. (1937) ,however ,showed no inhibition of somatic growth in the rat
when treated with large doses of testosterone although these doses were
sufficient to inhibit gonad development in both sexes.

The inhibition

^ of gonadal development can be explained through pituitary action; how­
ever, the discrepancy with Rubinstein’s work is yet to be explained.
Turner, et al. (1941), in agreement with McEuen's work, found no effect
of testosterone propionate in dosages as high as 2 mg. daily on body
weight increase or bone growth in castrated male rats even with pro­
longed injection from the day of birth.

Rubinstein’s group (1939a)

found a significantly lesser final body weight in the castrate male rat
and at the end of the experimental period the castrate group also showed
a smaller size, averaging 194 mm. in length as compared to 205 mm, in
the control group.

It was assumed that the animals were of equal size

at the start of the experiment and the rats were not measured; only
final measurements were made.

As indicated in a later part of this

thesis, the procedure for measuring animal lengths especially highly
active forms like rats and mice is subject to some variation and the

6

significance of the above measurements is to be questioned especially
where the assumption is made that the animals are of equal lengths at
the start of the experiment.

After later adjustments of dosage levels,

Rubinstein, et al. (1940) were able to stimulate the growth of castrate
rats.

The testosterone-treated castrate males showed an average dif­

ference of 12 mm; in body length above that of the castrate controls.
Ebchakian (1941) in a study of rates of absorption of testosterone
propionate pellets in mice, noticed that castrate controls accumulated
large amounts of abdominal fat while the implanted animals had only
traces.

The early work of Ebchakian and Uurlin (1935) with human pa­

tients stimulated Ebchakian to postulate protein anabolic effects of
testosterone propionate as a probability.

In a series of experiments,

he found that the administration of testosterone propionate to castrated
adult male rats produces a marked reduction in urinary nitrogen but in
about a week after the beginning of the injections the nitrogen excre­
tion gradually returns to normal in spite of repeated injections.

Af­

ter 30 days treatment with testosterone propionate, the mice average
3.4 gm. body weight more than that of the controls (Ebchakian, 1944a).
The observations by early workers of increase in body weight fol­
lowing small doses of testosterone propionate provoked the question as
to exactly what accounts for the Increase.

Is the increase in weight

due to the known growth effects on the accessory sex organs, an increase
in muscle mass, or an increase of all tissues generally?
analyzes the mechanism and probes for the site of action.

The question
Kenyon, et al.

(1940) state that the 102.7 to 252.2 gm. of protein estimated as being
retained by their men patients is not accounted for by increases in the
bulk of the genital tissue and represents deposits of new material

7

elsewhere la the body.

Koehakian (1944c) shewed a great Increase In

kidney size after steroid hormone treatment by pellet Implantation for
thirty days in mice and hamsters but not in guinea pigs.

Wrete (1945)

found no increase in rabbits, but Selye (1939) found kidney size increase
in testosterone propionate-treated mice.

Papanicolau and Ealk in 1938

made the preliminary observation of pronounced increase in size of the
temporal muscle of testosterone propionate-treated immature castrate
male guinea pigs over that of immature male controls.

Other muscles

of the body responded in the same way but the temporal muscle alone was
used quantitatively as indication of the response.

The above facts

provided good evidence that the protein anabolic effect of androgens
was not entirely on the accessory organs in spite of the fact that
great changes in weight do occur there.

The evidence of temporal

museleaffeet was confirmed in 1948 by Koehakian, et al., working with
short-haired English albino guinea pigs.

Castration decreased the

weight of the temporal muscles to less than one-third that of the nor­
mal animals.

Testosterone propionate administered as a pellet restored

the temporal muscles to about half normal size.

The castrated guinea

pigs did not gain as much weight as the normal controls.

The testos­

terone propionate (12.5 mg. /day) treated animals showed a 39$ gain in
body weight over that of the control animals.

The slight restoration

of temporal muscle indicated that maintenance rather than restoration
would be the better technique in indicating androgenic effect.

In a

later experiment, these workers castrated another group of guinea pigs
and immediately implanted them with pellets of testosterone propionate
of the same dosage as was used in previous attempts at restoration.
Thirty days later, the temporal muscles had been perfectly maintained.

8

These workers claim, however, that inasmuch as the steroid hormones
used were unable to really simulate the normal myotrophic and androgenic
status of the normal guinea pig, they are not "the hormone" of the
guinea pig or mouse testes.

The results to be described in the present

report show complete seminal vesicle-ventral prostate recovery or de­
velopment with a sufficient amount of the same hormone in mice.
Koehakian and Stettner (1948a), in work with mice, showed that the
change in body weight was due in part to changes in weight of the ac­
cessory sex organs and kidneys but that the greatest synthesis of pro­
tein occurred in the carcass.

The total amount of fat, however, de­

creased as the length of the treatment was increased showing an in­
creased catabolism of fat.
While attempting to find whether or not testosterone propionate
would hasten protein repletion after exhaustion of the reserve stores
by complete removal of food from adult rats until shortly before death,
Koehakian, et al. (1948) obtained supplementary evidence of the protein
anabolic effect.

Nine rats were castrated approximately 150 days before

the beginning of the fasting period.

At the end of 12 days, the five

animals used were given food plus 2.5 mg. testosterone propionate daily
as an injection while the remaining four animals had food alone.

There

was an initial spurt in body weight increase over that of the controls
but by the 31st day the control rats had attained the same weight as
the injected animals and continued to gain while the treated animals
were no longer or only slightly increasing their body weight.

During

the Initial spurt there was an extra nitrogen retention of 0.689 grams
or the equivalent of 20.7 grams of tissue.

9

Some of the same activities of testosterone propionate have been
shown in larger animals.

In 1935, Ebchakian, and Ebchakian and Murlin

"reported that 'male hormone' extracts prepared from medical student
urine produced a marked reduction in the urinary excretion of 'thin'
and 'fat' castrated dogs fed a constant diet" (cited by Koehakian,
1946b).

In 1936, Koehakian and Murlin produced the same results as

above with synthetic androstenedione.

Thorne and Harrop (1937) noted

decreased excretion of sodium and water in dogs following administra­
tion of testosterone.

Thorne and Engel (1938) noted a decrease in

urinary nitrogen in normal dogs given testosterone propionate.
Koehakian (1935) determined the effect of testosterone on protein
metabolism in castrate dogs and observed nitrogen retention in every
animal.

The nitrogen retention in each case was due to a decrease

in urinary nitrogen and not in fecal nitrogen.

The dogs also showed

a definite and gradual increase in body weight which continued for a
short period after injection and then gradually returned to basal.
Gaebler and Tarnowski in 1943 found that nitrogen storage was induced
in normal bitches by testosterone propionate.

Overbeek and Tausk (1946)

obtained regular increases in body weight of spayed monkeys with subse­
quent subcutaneous injection of 5 mg. testosterone propionate daily.
In the human being Sandiford and others (1941) found a significant
increase in creatinine excretion following 22 days of testosterone pro­
pionate treatment.

"They attributed this increase in creatinine excre­

tion to be a reflection of the development of a greater muscular mass'?
(cited by Koehakian, 1946b).

This diversion of protein, under the stimu­

lus of the potent steroid hormones, from catabolic to anabolic processes
deprives the organism of a small amount of energy.

There is, however,

10

no decrease in basal metabolic rate.

As a matter of fact, evidence

points to an increase in the basal metabolic rate (Kenyon, 1938;
McCullagh and Jones, 1942; and Bugbee and Simond, 1926),

Koehakian

(1946b) thinks that the energy to replace that lost to protein anabolism
and the increase in energy when it occurred was obtained by the utili­
zation of more fat.

Thorne and Engel (1938) obtained an increase in

nitrogen retention after treating a man having Addison’s disease with
25 mg. testosterone propionate daily for seven days.

Abels (1944)

treated normal and gastric cancer patients with testosterone propionate
and obtained both nitrogen retention and increased extracellular water.
The effects of male hormone in man have been deduced largely from
cases in which it was apparently absent such as in eunuchs, accidental
or therapeutic castration, or hypo-functional testicular conditions or
other abnormalities.

Gain in weight is found after treatment of eunuch­

oids with methyl testosterone (McCullagh and Rossmiller, 1941b).
McCullagh and McGurl (1940) state that weight gain during treatment of
eunuchoids with testosterone propionate is the rule.

Growth is affected

inasmuch as large dose3 of testosterone propionate can cause earlier epi­
physeal union than normally.

This can cause dwarfing.

However, Tinkler,

et al. (1944) show that testosterone has a tendency to accelerate longi­
tudinal bone growth in children without hastening epiphyseal union.

The

difference in results here may be due to the difference between children
and eunuchoids or it may be due to the dosage differences.

Dorff (1941)

in working with dwarfed infantile identical twins showed a rapid growth
in height and rapid gain in weight of one twin treated for a year with
chorionic gonadotropin.

The treated boy showed a linear growth spurt

of 4 inches as compared to the identical control twin’s growth of 1 1/4

11

inches.

The treated twin gained 14 lbe. in one year as compared to a

gain of 6 lbs. of the identical twin control.

Here nature has set up

the basis for a controlled experiment the likes of which a m not found
often in clinical medicine.

Presumably the chorionic gonadotropin was

low in folliole-stlmulating hormone and high in luteinizing hormone and
undoubtedly the amount of luteinizing hormone was enough to produce
interstitial cell stimulation in the testes as a spurt in development
of secondary sez characteristics was noticed in the treated twin.
Browne and Boss added evidence to the above when they showed that the
rate of growth of stunted children was more than doubled by treatment
with testosterone propionate (1941).
Tinkler, et al. (1942) treated 15 children with testosterone pro­
pionate and found an advance in growth rate of 13 of them.

Webster and

Hoskins (1940) administered 75 to 125 mg. testosterone to 8 hypogonadal
boys between 9 and 13 years of age and produced an increase in average
growth rate from 1.36 cm. to 3.6 cm. in 100 days.
In children suffering from Progeria, a disease with such strange
symptoms as extreme immaturity upon whieh has descended the blight of
premature senility including cessation of growth, Talbot, et al. (1945)
gave daily intramuscular injections of testosterone propionate and evidenoed a marked gain in body weight of 500 gm. per year.

Closely akin

to this, are the results of Kenyon, et al. (1942) who produced a
nitrogen retention in two 76-year-old men, approximating that of nor­
mal young men, by administration of 25 mg. testosterone propionate daily.
The same group produoed a uniform gain in body weight of from 1 to 2.6
kilograms per 10 day period when eunuchoids were treated with the same
dosage as above.

In accord with the above is the work of Wilkins and

12

Tleischman (1946a) who treated immature boys with testosterone and
observed protein anabolle changes.

Shelton, et al. (1947) set up an

experiment with four sets of premature twins, where one of each set
was treated with testosterone propionate intragluteally.

A distinct

shortening of time required to regain the birth weight and to gain
2500 grams was noted in the twins receiving treatment.

Somewhat the

same type of experiment as those indicated above "has been carried out
by nature in cases of interstitial cell tumors of the testicle which
cause marked somatic as well as sexual stimulation" (Albright, 1942-43).
This is well illustrated in a case reported by Rolands, et al. (1929)
of a boy at age nine who had the sexual and somatic development of an
adult man.

In a personal communication to Albright (1942) Browne made

some observations on a patient with such an interstitial cell cancer
with extensive metastases; the most striking findings were a 17-ketosteroid excretion of over 1000 mg./24 hours, and the failure of the
patient right up to the time of death to lose weight or to became de­
bilitated in spite of extensive metastases.

The large 17-ketosteroid

figure here would compare to large doses of male hormones given in
previously described cases where a weight loss was the result - this
discrepancy invites further question.

Albright and his group carried

out similar studies of the effect of testosterone propionate in dwarfs
suffering from panhypopituitarism.

The dwarfs grow in every aspect;

not only do their muscles and epiphyseal cartilages grow, but the en­
tire body grows.

Albright believes that testosterone stimulates pro­

duction of tissues rather than promoting the storage of nitrogen in
some other form (e.g. deposit protein).

He shews that from a clinical

point of view the findings in the adreno-genital syndrome (associated

13

with a cancer or adenoma of one adrenal cortex or with hyperplasia of
both adrenal cortices) are all consistent with the thesis that some hor­
mone is being produced in excess which has an action very similar to
testosterone propionate.

The 17-ketosterold excretions seem to be in­

variably high (60 mg./24 hours in many cases).

Such patients show

evidence of excessive somatic and sexual development in the male direc­
tion.

Thus the evidence herein submitted leaves little doubt as to the

protein anabolic action of the male hormone.

The thyroid hormone like­

wise in proper dosage is shown to act in a similar manner.

Growth Effects of Thyroid Hormone
The growth effect of the thyroid hormone has been known for many
years and extensive reviews in the literature make full discussion here
unnecessary (Hoskins, 1916; Cameron and Caxmleheal, 1920; Schneider,
1939; and Roger and Turner, 1943).

Deprivation of thyroid hormone In

immature animals by thyroidectomy or with thiouracil, the newer ap­
proach, results in growth stasis.
osseous development.

Examination of bones shows delayed

In rabbits thyroldectomized at an early age,

growth is definitely inhibited compared to that of litter mate con­
trols,

C.D. Turner shows pictures of such rabbits in his text (1949),

The normal controls weighed 1600 grams compared to the thyroldectomized
animals which weighed only 800 grams.

Scow and Simpson (1945) in thy-

roidectomized newborn rats showed exceedingly slow but continuous in­
crease in body weight and size of skeleton.

Gonads and dependent ac­

cessories were markedly subnormal in development.

The cretinous human

being shows what happens to growth of an individual when the thyroid is
not secreting sufficient quantities of its hormone.

In addition, the

14

clinical evidence of thyroid treatment of young cretins is sufficient
evidence of the anabolic effects of the thyroid hormone.

Topper and

Cohen (1928) obtained in a relatively short time an extraordinary in­
crease in growth of four normal children 10 to 12 years old,

Further

evidence is presented by G. B. Dorff (1935) who stimulated the growth
of small but apparently normal children 6 to 8 years of age by daily
doses of from 1 to 5 grains of thyroid substance although Johnston
(1941) states that more than one grain of Parke-Davis thyroid would be
catabolic in its reaction.
Evidence of overgrowth in cases of juvenile thyrotoxic patients
is so clear and occurs with such remarkable regularity that Hertz and
Galli-Mainini (1941) consider it as one of the cardinal symptomatic
manifestations of the disease in this age group.

wIts growth effect

(thyroid) can be considered as truly synergistic to the normally exist­
ing factors for structural increase (substrate)".
the same.

Staith (1933) reports

Koger, et al, (1942) show that thyroxine-treated female al­

bino mice repeatedly gained an average of 28# more weight during a
period of five weeks than did the controls.

They reported that most

investigators attribute a catabolic effect to thyroid treatment as in­
dicated by body weight but that Moussu (1899) and Dott (1923) obtained
acceleration of the growth rate in feeding small amounts of thyroid
tissue to immature dogs.

Schafer (1912) and Hoskins (1916) both re­

ported increased rate of growth and feed intake in female rats fed
small amounts of thyroid.

Robertson (1928) reported results in mice

which were substantiated by Koger, et al. above.

The amounts of thy­

roxine used by Koger were very small - 0.015 to 0.04 mg. daily by sub­
cutaneous injection.

More recently Koger, Reineke and Turner (1943)

15

hare reported the influence of thyroactive iodocasein on growth.

The

thyroactive iodocasein-fed mice gained from 16 to 23% more in weight
and an average of 28% more in length than the untreated controls.
Heineke, et al., (1948) showed definite growth stimulation of thyroprotein on several strains of pigs.
Evidence somewhat contradictory at first glance is shown in the
early experiments of Gudernataoh (1914) where he found that rapid
metamorphosis of frog tadpoles occurred when desiccated thyroid is
sprinkled into the water.

Although he says growth is retarded while

differentiation is greatly accelerated it is clear that he was using
the term growth in the limited sense referring to Somatic growth.
Two points occur to the reader here, however, (1) the developmental
process unique to the frog is such that the loss of a long tail would
hardly warrant growth (linear) effects, and (2), the excessive matura­
tion is again not conducive to size gains.

Assimilar situation is

present in rapid cleavage divisions of fertilized mammalian eggs where
size remains the same; the tadpole compares favorably with the embry­
onic stage of mammals.

After the process of differentiation has slowed

up, one would predict a rapid growth in thyroid-treated animals, however
the facts reveal that tadpoles invariably die after this treatment.
Alexis Carrell (1913) was able to increase the growth of brain
tissue several fold by the in vitro addition of thyroid substance,
thus shedding a little light on the site of action of the thyroid ana­
bolic effect.

The exact site and mode of action of the thyroid hormone

are still questions being energetically investigated and as will be
seen, furnish indirect evidence to its anabolic effects.

Leblond (1949)

shows distribution of a physiological dose of radiothyroxine in the rat

16

to be concentrated mainly in liver, gastrointestinal tract, muscles
and skin in studies made two hours after injection.

Although great

emphasis was placed on large concentrations in liver and gastrointes­
tinal tract, little was made of the fact of heavy concentrations in
the muscles; confirmatory evidence for anabolic activity.

Dr. Albert

Bitzmann (1949) in commenting ott Leblond's paper presented to the New
York Academy of Sciences, relates Leblond*s findings with his own
clinical work.

Interpreted in one way "patients with marked hyperthy­

roidism, in diffuse toxic goiter, the abnormal or exaggerated findings
of excess skin perspiration, irritable heart, diarrhea, altered liver
function and aimless exaggerated muscle movements link up with those
organs of tissue systems in which the thyroxine content is high.

Inter­

preted in another way, Leblond's findings show the association of high
thyroxine concentration in tissues or organs where functional need and
cell metabolism are greatest".

Addis, et al. (1938) show the protein

content of heart, kidney and liver to increase in thyroxine-injected
rats.

This ties in very well withlRitzmann's results and agrees with

his interpretation.
Research workers began inquiring into the relationships of the
other endocrine glands to the testis after clear-cut castration effects
were noticed in the pituitary.

Castration causes vacuolization of
4

basophil cells in the anterior pituitary.

Andersen and Kennedy (1933)

found atrophy of the thyroid three weeks after castration of a number
of rats while consistent atrophy of the thyroid is found in all cases
eight weeks after castration.

Further evidence from Carriere, et al.

and Nathanson, et al. (cited by Jones, et al. 1941) show that hypersecre­
tion of the thyroids occurs with treatment with testosterone propionate.

17

The histologic changes in the thyroid include increase in cell height,
increase in number of mitotic figures, assumption of a round shape by
the nucleus, migration of the nucleus toward the base of the cell and
finally vacuolization of the colloid.

Effects of Combinations of Male and Thyroid Hormones
At this date, no large scale direct study on the relationship of
the two hormones has been undertaken nor has anyone done any quanti­
tative work with testosterone on immature laboratory animals.
r

The relationship of testosterone propionate with the growth hor­
mone of the pituitary has been studied in male rats by Simpson, et al.
(1944) who found what they called a synergistic growth effect.

This

work was repeated by Koehakian and Stettner in 1948 (a) on male mice
and the results of Simpson were corroborated.

Thirty days after treat­

ment with a 14 mg. pellet of testosterone, albino male mice gained on
the average

of 2 1/2 gm. more than the

with pituitary growth hormone

gained 3

controls.Those animals treated
gm. more than the controls.

The

combination of pituitary growth hormone and testosterone propionate gave
a 5 1/2 gm.

increase over the controls

- an exactsummation effect.

The authors

state that "It is unlikely

that theseresults are due to a

further stimulation of the same intermediary metabolic processes for
doubling the dose of testosterone does not produce a further increase
in the body v/eight of the castrated mouse".
Tive different groups of workers, however, have carried out in­
directly some aspects of this experiment in their handling of clinical
cases.

Albright, et al. (1941) had a patient with panhypopituitarism

on thyroid therapy for six years.

The patient showed increased weight

18

and height.

However, after testosterone propionate therapy in addition

to the thyroid therapy, there was a marked weight and height increase
for the next three years.
Eidelsberg and Ornstein (1940) noticed that the effect of adminis­
tration of 3 to 5 grains daily of thyroid on the basal metabolic rate
of human young males was more marked during the periods of administra­
tion of testosterone propionate.

Also, Klnsel, et al. (1944) found

that testosterone propionate in thyrotoxic patients induces a strongly
positive nitrogen balance and gain in weight even when the caloric in­
take is less than the caloric expenditure - an action indicated a year
earlier by the same group (Beifenstein, et al. 1943).

Thyrotoxicosis

is characterized by an increase in the urinary excretion of nitrogen
and creatine and by a decrease in body weight.

Testosterone propionate

in small doses had the opposite effect on these three variables.
Armstrong (1944) working with a patient with both diabetes mellitus
and Addison’s disease gave 0.032 gm. thyroid daily (about 1/2 grain)
in addition to testosterone propionate for three successive days and
noticed no change in body weight but he reported that the patient ex­
hibited a remarkable increase invigor, well-being and appetite.
data, however, are Insufficient to show any real trend.

The

Wilkins (1945)

treated a 4 1/2 year old hypothyroidboy with thyroid, and later with
thyroid plus methyl testosterone but showed no gain in weight in this
one case.
From the facts disclosed in the foregoing review, it seemed ob­
vious that both testis hormone and thyroid hormone will influence pro­
tein metabolism under certain conditions.

Bather fragmentary clinical

data indicate that there may be some relationship between the two hormones

19

In their effect on protein anabolism.

No controlled experiments have

been found bearing specifically on this problem.

The experiments to be

reported were designed to determine more fully, the effects of the two
hormones given alone and in combination on growth and protein metabolism.

EXPERIMENTAL PROCEDURE AND RESULTS

Preliminary Investigation
In order to determine the amount of male hormone necessary to
maintain the sex accessories in this particular strain of mice the
following preliminary assay was carried out.

Young male albino mice

of the Rockland Strain and bred in our laboratories were divided into
four groups of five mice per group (mice averaged 10 to 16 gms. weight).
Fifteen animals were castrated and given subcutaneous injections of
testosterone propionate in varying dosages in corn oil daily for 5
days.

The results showed that none of the dosages used brought

seminal vesicle and ventral prostate weights up to those of normal
controls.

The average combined weights of these organs are shown

in Table I along with the particular dosage used.

Evidence in the

literature testifies to the fact that testosterone propionate even
in tremendous dosage rarely maintains seminal vesicle weights up to
normal although in pellet form the sex accessories were maintained
up to normal with certain doses.

The assay, however, primarily Indicated

the approximate amount of hormone to be used in the later phases of this
study and to develop a range of dosages around this point for young ani­
mals.

Greene and Burrill (1941) found the sex accessory response to be

much less variable in young rats than in older ones.
Following this preliminary assay, the first of four major experi­
ments was started.

The first experiment was done to establish the pro­

cedure, to learn the techniques inherent in this type of procedure, to
test the efficiency of pellets as compared to injections, to determine
quantities of food and water used under the regime, and to evaluate

20

21

TABLE I
FIVE DAY PRELIMINARY ASSAY 0? TESTOSTERONE PROPIONATE
ON CASTRATE MALE MICE
Group

A

Dally Dosage
Testosterone Propionate
50

Seminal Vesicle-Ventral
Prostate Wt./25 Gm. Body Wt.

yg.

63.0 mg.
±3.1*

B

150 Jig.

65.3 mg.
±3.9

C

300 jig.

76.4 mg.
±4.1

D

Normal! littermate
controls

119.2 mg.
±13.0

Five mice were used in each group.
♦Standard error.

22

responses to the dosages of hormone used - in short, to orient the
problem.

General Procedure
The albino mouse was used as the experimental animal because
numerous investigations have shown it to respond favorably to suit­
able thyroid dosage.

The mice were males of the Rockland Farm strain.

In each of the 4 experiments, the mice were castrated three days
after weaning.

All animals were fed a Purina diet1 ad libitum which

came as a pellet and was ground into a coarse powder.

The food was

placed in specially designed food boxes which kept food waste at a
minimum.

Wherever thyroprotein was part of the treatment, Protamone

(an iodlnated casein compound supplied by the Cerophyll Laboratories;
prepared by the method of Reineke) was mixed thoroughly into the diet
in the desired percentage.

The synthesized compound resembles the

thyroid hormone in its physiological activity and had both biological
and chemical assays made on its thyroxine content as a check on its
aotivity, uniformity and reliability8.
In all cases where the animal was to receive testosterone propio­
nate treatment by pellet technique, the pellet was implanted under the
skin of the neck through a small Incision.

This was done shortly after

^Purina Laboratory chow pellets consist of: Meat meal, dried skimmed
milk, wheat germ, fish meal, liver meal, dried beet pulp, corn grits,
oat middlings, soybean oil meal, dried alfalfa meal, molasses, ribo­
flavin supplement, brewers dried yeast, thiamin, niacin, Vitamin A
and D feeding oils, D activated plant sterol, 1# steamed bone meal,
iodized salt and MnSO^.
8See Vitamins and Hormones. Vol. IV, (1946) for full description of
history and technique of Reineke*s synthesis.

23

the castration operation.

The pellet was pushed about an inch from the

site of the incision with a very narrow forceps.

A single stitch closed

the incision and the area was painted with Mercresin.

The particular

font of male hormone used was testosterone propionate because of its
efficiency of absorption in subcutaneous areas (Leathern, 1948) and its
effectiveness.

The results of Parkes (1936) showed testosterone pro­

pionate injections to be more effective than testosterone acetate which
in turn was more effective than the free hormone.

A year later, Deansely

and Parkes found that pure crystalline androgens were extremely effec­
tive when implanted subeutaneously as tablets.

They obtained the usual

response - the enlargement of the prostates and seminal vesicles in
castrate male rats.

In 1941, Biskind and Heyer verified the above ex­

periment and obtained effective androgenic response from immature cas­
trate rats with pellets of testosterone of about 4 mg. implanted subcutaneously in the back of the animal.

These workers showed that the

hormone maintained the same degree of activity when compared to oil in­
jections,

"This uniform rate of absorption from pellets may permit the

concentration of the androgen in the tissues to be maintained at a con­
stant level, whereas with oil injections absorption may be rapid and
uneven, producing variations in the concentration of the injected andro­
gen. "
Kochakian in 1943 used the pellet techniques extensively in his
steroid hormone work on guinea pigs, rats, mice and men.

Tests, et al,

(1940) used the pellet technique successfully in the monkey and estab­
lished average absorption rates on the basis of accessory organ weights.
More recently Vills, et al. (1949) state that the variance of the assay
decreases when you go from 0.2 cc. subcutaneous dose given once daily

24

to a 0.1 eo. subcutaneous dose given twice daily.

This leads one to

believe that greater accuracy is obtained from smaller uniform doses
which would come from gradual pellet absorption.
The decision to use the pellet technique was made on the basis of
the above advantages including the time-saving advantage of eliminating
one or two daily injections into sixty animals for a period as long as
35 days.

The technique also reduces the amount of handling of animals.

Regular records were taken of body weight, and food and water con­
sumption during the course of each experiment.

The animals were kept

in cages on steel racks in an air-conditioned laboratory under uniform
conditions of lighting and humidity.

At the end of each experiment,

the mice were sacrificed, body weights taken and in the first two ex­
periments their final lengths determined.

The seminal vesicles and

ventral prostates were removed carefully from each mouse, trimmed of
extraneous tissue, and weighed rapidly on a Roller-Smith torsion balanee.

These glands were then placed under the peritoneum and saved for

later analysis.

The animals treated with testosterone propionate pel­

lets had their necks explored and the pellets recovered.

These pellets

were washed several times in distilled water and thoroughly dried in a
desiccator to constant weight.

The weights were recorded and compared

to the original pellet weights in the particular animal.

Each animal

was placed in a specimen jar, sealed tightly and stored in a refrigera­
tor in a frozen condition.
Experiment One
In the first experiment, testosterone propionate pellets.were made
by cutting large tablets into smaller ones by running a hot tungsten
wire down through the tablet and then touching the hot wire to the edges

25

of the small pellet to smooth it into a sphere.
and the most uniform pellets selected for use.

These were then weighed
The pellet weights from

this method of manufacture ranged from 10 to 16 mg.
The mice of each group in the experiment were kept together in one
large cage and individual mice were identified by the customary ear mark­
ings.

The castrated animals had their testes removed through a small ab­

dominal opening and the incision was closed with silk sutures.

One stitch

was taken in the peritoneum and one or two stitches in the muscle and skin
depending on the size of the Incision.

The area was painted with Mer-

cresin (a highly germicidal product of Upjohn Co.), body weights recorded,
length measured and the animal was returned to the cage.
ments of length were made:

Two measure­

one was from the tip of the nose to the end of

the rump; the other was made from the tip of the nose to the end of the
tall.

While the animal was still under the effects of ether, the back of

the animal was pressed down gently and the measurements of length made.
Those animals in the testosterone propionate treated group had
pellets inserted in the nape of the neck.
operative recovery.

Two days were allowed for

The animals were then placed in their proper groups

and put on the detlred food regime.
The mice in this experiment were grouped according to the outline
Indicated in Table II.

The average gain in weight per week far each of

five consecutive weeks is lndieated in Table II.

The average cumula­

tive weight gains of the various groups are plotted against time as the
abscissa and illustrated in figures 1 and 2.
The first noticeable relationship is the effect of thyroprotein.
Those groups which had thyroprotein in the feed showed definite drops
in the growth curves between the 4th and 5th weeks of the experiment

26

TABLE II

AVERAGE CUMULATIVE WEIGHT GAINS OF MICE IN EXPERIMENT ONE
Experimental Plan
No.
Group Ani­
mals

Description

Gain
by
First
Week
in
Grams

Gain
by
Second
Week
in
Grams

Gain
by
Third
Week
in
Grams

Gain
by
Fourth
Week
in
Grams

Gain
by
Fifth
Week
in
Grams

1A

7

Castrate controls

6.5

8.3

U.8

13.2

14.5
±1.0*

IB

8

Castrates plus
thyroprotein
(0.196)

6.6

9.9

11.3

11.5

10.3
±1.5

7.6

9.7

11.0

12.9

14.8
±0.8

6.7

9.2

12.2

13.6

9.7
±1.1

1C

133

8

8

Castrates plus
testosterone
propionate
(12.5 mg.)
Castrates plus
thyroprotein
(0.1$) plus
testosterone
propionate
(12.5 mg.)

IE

6

Normal controls

5.7

9.2

11.9

13.6

14.5
±2.5

IF

5

Normals plus
thyroprotein
(0.1$)

5.8

10.2

15.2

18.5

17.5
±2.6

♦Standard error.

27

IA
IB
1C
■ ID

CASTRATE CONTROLS
CASTRATES+ T.R (0.1%)
CASTRATES + T.PROR
CASTRATES + T.PROR
+ TP (0.1%)

AVE. CUMULATIVE

WEIGHT

GAIN

(GMS.)

’

IE NORMAL CONTROLS'
IF NORMALS tT.R (0.1%)
figure 2

figure

2

3

4

5

WEEKS

Figures 1 and 2.
Week Period.

Growth Curves of Mice in Experiment One Over a Five

T. R = thyroprotein.
T. PROP. = testosterone propionate.

£8

compared to the other groups.

This indicated that the level of thyro­

protein in the food mas too high and mas causing a marked hyperthyroid
condition mith the concomitant loss in weight.

The indication mas to

loner the thyroprotein level for the next experiment.

In the uncas­

trate animals receiving thyroprotein however, the early increase in
body meight mas so large, that at the end of the fifth meek, the body
melghts mere still significantly higher (F ■ 2.37, see methods in
Table 1 of the Appendix) than those of the normal controls in spite
of the drop in the last meek.

Group ID (castrates treated mith both

testosterone propionate and thyroprotein) mas off to a slow start but
finally rose above the other castrate groups.

The rise mas above that

of group 1C (castrates plus testosterone propionate alone) but the
amount mas not significantly different.

At the end of the fourth meek,

the order of body meight increase from highest to lowest was:
1A and IB.

ID, 1C,

From the known growth effects of thyroprotein it mas clear

that a lowered thyroprotein dosage mould have reversed the positions
of IA and IB,

This mas verified in the second experiment.

A study of the dally consumption of food and mater mas made for
each group and the results from the data are illustrated in Table III
following.

The results reveal increased food consumption by the thyro­

protein groups over and above that of the other groups.

The normal

castrate controls (1A) averaged 3.8 gms. of food per mouse per day
whereas the castrates plus thyroprotein animals (IB) averaged 4.7 grams.
The castrates plus testosterone propionate (1C) averaged about 4 gms.
daily while the castrates plus testosterone propionate plus thyroprotein
(ID) ate about 5 gms. each.

It appears that 0.1# thyroprotein in the

feed increased daily food consumption by roughly 1 gm. per mouse daily.

29

There were no significant differences in the food consumption of the
respective groups of mice that can he attributed to the treatment with
testosterone propionate,

Among the uncastrated control animals, the

normal controls (17) again consumed about a gram less food than the
normal animals with thyroprotein.
The consumption of water (Table III) follows the same pattern as
the consumption of food in the various groups.

Castrate controls

drink*- on the average of 7.1 gms. water per mouse daily while the
castrates plus thyroprotein drink 9.4 gms. - a difference of 2.3 gms.
per mouse per day.

This greater consumption of water is found wher­

ever thyroprotein (0.1J&) is contained in the diet.
The average mouse consumed a total amount of food and water for
the five week period as indicated in Table 17.

The food intake per

unit weight gain is a criterion of "efficiency"; the term as used in
this paper refers to body economics and not to energy as used by Brody
(1945) and others.

The last two columns of Table 17 show this "ef­

ficiency" for food and water respectively.

Analysis of the data shows

groups A and C as the most "efficient" groups of animals, that is, the
least amount of food was consumed per unit weight gain.

The groups

receiving thyroprotein at the level indicated, i.e., groups B, D and 7
were the least "efficient".

The administration of testosterone pro­

pionate apparently has no effect on food intake except perhaps a slight

*-The normal castrated and uncastrated animals follow Adolph's (1949)
rate of water intake to body weight equation: I ■ aB^
I » rate of water intake in gms./hr.
a * constant 0.01
B * body weight in gms.
k « exponent 0.88

30

TABLE III
AVERAGE WATER AND FOOD CONSUMPTION/MOUSE/DAY
LA

Weak

IB

1C

ID

IE

IF

Castrates
Castrates Castrates
Normals
+ Thyro­
+
Castrate + Thyro­
protein
Normal + Thyro­
Controls protein Test. Prop.
Controls protein
(0.1#) +
(0.1#)
(12.5 mg.) Test. Prop.
(0.1#)
(12.5 mg.)
Water Consumption (in Grams)

1

5.8

7.1

6.6

7.6

7.6

7.9

2

6.9

10.1

8.6

10.2

9.2

11.0

3

7.2

10.6

7.8

9.7

8.8

12.6

4

7.9

9.9

8.1

10.5

9.2

13.8

5

7.7

9.2

8.4

11.1

9.1

13.1

7.1

9.4

7.9

9.6

8.9

11.7

Aye.

Food Consumption (in Grams)
1

3.2

3.6

3.2

3.8

3.9

3.4

2

3.7

4.9

4.0

4.8

4.6

5.0

3

3.9

5.0

3.8

5.2

4.9

7.2

4

4.3

5.0

4.2

5.7

4.4

6.3

5

4.1

4.9

4.2

5.0

5.0

6.6

3.8

4.7

3.9

4.9

4.6

5.7

Aye.

31

TABLE IV
AVERAGE POOD AND WATER-BODY WEIGHT GAIN RELATIONSHIPS PER MOUSE
(EXPERIMENT ONE)
Five
Five
Weeks Weeks
Total Total
Pood Water
in
in
Grams Grams

Average
Food
Five Wk. Intake
Gain in Per Unit
Body Wt. Wt. Gain
in
in
Grams
Grams

Water
Intake
Per Unit
Wt. Gain
in
Grams

Group

Description

1A

Castrate controls

133.9

249.5

14.5

9.2

18.3

IB

Castrates plus
thyroprotein
(O.l#)

163.6

329.6

10.3

15.9

32.0

Castrates plus
testosterone
propionate
(12.5 mg.)

135.5

277.1

14.8

9.2

18.7

Castrates plus
thyroprotein
(0.1#) plus
testosterone
propionate
(12.5 mg.)

171.7

343.9

9.0

19.8

38.2

IE

Normal controls

160.0

307.6

14.5

11.0

21.2

IP

Normals plus
thyroprotein
(0.1#)

200.7

410.9

17.5

11.6

23.5

1C

ID

32

synergistic effect when administered simultaneously with thyroprotein.
Group D consumed more food per unit weight gain than group B.

This is

not shown by the uncastrated controls (17) receiving thyroprotein.
The water consumption per unit weight gain shows exactly the same
picture as in food consumption.

Again 1A and 1C are about equivalent

and more "efficient4* than the other groups.

Group IB is slightly

higher than IB, again indicating the synergistic action mentioned
above.

The groups receiving thyroprotein were in every case higher

than the other animals.

The parallelism of food and water consumption

lends credence to the data obtained.

The castrate animals treated with

testosterone propionate (1C) gained more weight with less food and water
than the normal controls (IB).
The data on body lengths included measurements from nose to rump
and from nose to end of tail.
Table Y below.

These growth changes are presented in

Considering the evidence of McMeekan (1940) and Wallace

(1948), and following the formulations of Huxley concerning the existence of growth gradients, one can analyze the growth data from the
gradient aspect.

Thus the ratio of nose to rump increase over nose to

end of tail increase is used with the data.

The changes in length

shown took place over the period of the experiment (35 days).

Maroney

and Johnston (1939) consider height data reliable only if taken over
long periods of time.

It is to be noted that groups B and B with thyro­

protein treatment have lower ratios as indicated in the table which
shows greater effect of thyroprotein on tail growth than on "body"
growth.

This can be understood when we recognize that the posterior

parts of a growing organism are in a less differentiated state and
therefore more plastic.

These results are in agreement with McMeekan

33

TABLE 7
FIVE WEEK INCREASE IN BODY LENGTH AND SEX ACCESSORIES
(EXPERIMENT ONE)

Group

Description

(A)
(B)
Average Average
Ave. Wt.
Increase Increase
Sem.Ves.Nose to Nose to Ratio ^ Ventral
Rump
End of
Prostate
Tail

Ave. Wt.
Sem.Ves.Ventral
Prostate
Per 25 Cta.
Body Wt.

1A

Castrate
controls

27 mm.
±1*

51 mm.
±2

0.53

14.2 mg.
±4.9

IB

Castrates plus
thyroprotein
(0.1#)

25
±1

51
±2

0.50

5.0
±2.4

Castrates plus
testosterone
propionate
(12.5 mg.)

29
±1

52
±2

0.56

196.4
±16.3

171.1

ID Castrates plus
thyroprotein
(0.1#) plus
testosterone
propionate
(12.5 mg.)

26
±1

47
±2

0.55

146.3
±7.3

152.4

Normal
controls

24
±3

45
±7

0.53

165.0
±11.4

125.0

IF Normals plus
thyroprotein
(0.1#)

32
±4

56
±9

0.57

104.5
±13.4

82.4

1C

IE

♦Standard error.

12.8

5.3

34
(1940) who found that the sacral bones of the vertebral column In pigs
show the greatest growth from birth to 88 weeks of age and that the
lumbar, thoracic, cervical and skull bones follow in order.
Inasmuch as a criterion is needed to substantiate the androgenic
effect, the sex accessories serve in this capacity here and so the
seminal vesicles and ventral prostates were carefully removed at the
end of the experimental period and their weights recorded (Korenehevsky,
et al., 1932, and Price, 1944).

In the event that a particular animal

treated with testosterone propionate had only negligible seminal vesicle
and prostate weights, the animal was considered as not under the effect
of the hormone and stricken from the records.

In the few cases where

this was found, a check revealed that no pellet was recovered from the
animal.

The pellets evidently slipped out of the Incision and were not

in the animal during the experimental period.

The averages of the acces­

sory sex organ weights are shown in Table V for each of the groups.
Figures on the basis of 85 gm. body weight are also given.

Deansley

and Farkes (1933) find high correlation of body weight to sex acces­
sories in the 19 to 24 gm. body weight range in mice.
The data indicate typical castration effects in groups A and B
where no androgenic hormone was given to the mice.

Groups G and D

showed typical maintenance of the sex accessories.

In castrate mice

treated with testosterone propionate and thyroprotein and in the nor­
mal animals treated with thyroprotein there is apparently a detrimental
effect of the thyroprotein on the sex accessories.

This adds weight to

the hypothesis stated earlier that the level of thyroprotein was too
high in this first experiment and that it was having a definite cata­
bolic action throughout the body.

The greater average weights of the

35
sex accessories in group 1C compared to the normal controls (IS) is
either due to the wide range of the accessory weights even in normal
animals, the few animals used in this first experiment, or damaging
dosage of testosterone propionate.
The results of this first experiment seem to indicate then, that
the dosage levels of both testosterone propionate and thyroprotein are
above that which could be expected to give anabolic effects.

Having

roughly established the above, the next phase of the research was set
up with more appropriate dosage levels and with larger numbers of ani­
mals.

The results of the first experiment must be considered tentative

and of orientation value.

Experiment Two
The second experiment of this problem followed the first closely
in time.

Slightly older mice were used here as compared to the first

experiment.

The average starting weight of the mice was 18.5 gas.

The

mice were relegated to the various groups so as to make fairly uniform
experimental conditions.

The experimental plan is shown in Table VI.

The castrations of groups 2A, 2B, 2C, and 2D were done over a
period of 48 hours and in the same manner as in experiment one.

The

male hormone pellets were placed in the nape of the neck on the day
following castration.

Two days were allowed for operative recovery and

the animals were placed, one group to a large cage, with proper food
and water.

In this experiment the thyroprotein-treated mice had

Protamone mixed in their feed on the basis of 0.05# of the feed.

The

experiment was carried out for a five week period during which time
daily records of food and water consumption were made.

The weights of

36
the animals were taken weekly.

A measurement of length was made on the

first day of the experiment and on the 35th day.

At the end of the ex­

periment, the animals were treated as previously indicated in experi­
ment one.
The results of analysis of the comparative gain in weight of the
various groups of animals is compiled in Table VI below.
Translation of the data in Table VI into curves appears inFigures
3, 4, 5, and 6.

In Figure 3, one notices the growth effect of thyro­

protein at the 0.0556 level on castrate mice as compared to the castrate
controls.

Likewise, in Figure 4, a comparison of normal controls with

normal animals treated with thyroprotein shows a definite growth stimu­
lus of thyroprotein.

Analysis of Figure 5 reveals that the addition of

testosterone propionate has little effect on the growth rate at the
level of hormone used here (10.2 mg. pellet).

Apparently the dosage of

testosterone propionate is still too high (the pellets averaged 12.5
mg. in the first experiment) and is having a detrimental effect on the
animals.

Figure 6 shows that the additional treatment with testosterone

propionate of castrate animals receiving thyroprotein has a slight
growth-inhibiting effect.

A number of workers (Korenchevsky, et al.,

1937) have shown that there is a body weight-depressing action of
large quantities of testosterone propionate.
Every one of the curves shows a drop in weight during the fifth
week of the experiment - a result which cannot as yet be accounted for
in the experimental conditions.

For this reason, cumulative weight

gains at the end of four weeks should be examined and compared.

The pre­

diction after the first experiment that a lowered thyroprotein level would
result in better weight gains is verified here.

The level of thyroprotein

in experiment two was half of that used in experiment one.

TABLE VI
AVERAGE CUMULATIVE WEIGHT GAINS OF MICE IN EXPERIMENT TWO

Experimental Flan

Group

No.
Ani­
mals

2A

15

Castrate
controls

2B

14

Castrates plus
thyroprotein
(0.0556)

2C

2D

14

14

Description

Castrates plus
testosterone
propionate
(10.2 mg.)
Castrates plus
thyroprotein
(0.05%) plus
testosterone
propionate
(10.2 mg.)

BE

16

Normal
controls

2F

14

Normals plus
thyroprotein
(0.05%)

*Standard error.

Gain
by
First
Week
in
Grams

Gain
by
Seeond
Week
in
Grams

Gain
by
Third
Week
in
Grams

Gain
by
Fourth
Week
in
Grams

Gain
by
Fifth
Week
in
Grams

5.0

8.5

11.3

11.0

10.3
±.7*

5.5

9.2

12.4

13.5

12.5
±.8

5.0

8.0

9.9

11.7

10.6
±1.1

6.5

9.4

11.6

12.8

8.2
±.8

6.2

8.8

10.0

10.8

8.6
±.5

5.9

9.7

11.0

12.2

9.3
±.7

38

2E NORMAL CONTROLS
2F NORMALS + T.R (05%)

2A CASTRATE CONTROLS
26 CASTRATES + TP. (09%)

m

tigura 3

'

I ' 1~l ' 1 I 1 ' I
2A CASTRATE CONTROLS
2C CASTRATES + T.PROR (KJ2MGJ
2 0 CASTRATES ♦ T.PROP. 0Q2MGJ
+ T.fi(03%)

ftgur« 4

1—1 r ■ ■ i ■ ■ i ■

I

1

'

I

2 6 CASTRATES ♦ T.R (03%)
2 0 CASTRATES + T.PROR Q02MG)
+ T.R (03% )'

u8

flflur* 6

WEEKS

Figures 3, 4, 5 and 6.
Over a Five Week Period.

WEEKS

Growth Curves of Mice in Experiment Two

39
A comparison of the daily consumption of food and water for the
various groups reveals further points of interest.

The results are ex­

pressed in Table 711.
The effect of thyroprotein in the diet is again clearly indicated
in the dally food consumption of groups 2Bt 2D, and 2F.

The thyro­

protein treatment caused an Increased food consumption of 1 gm./day
more than in those animals not receiving thyroprotein.

As in experi­

ment one, the animals treated with testosterone propionate show no
significant difference in food consumption from that of the castrate
controls.
tion.

The water consumption follows the pattern of food consump­

Large amounts of water are consumed by the groups which had

Protamone added to the feed.

See Table VII,

In every case, water and food consumption increased slowly with
maturity as would be expected.

This same trend was seen in experiment

one.
The "efficiency" of the groups of mice in converting food into
body weight is again of special interest.

The food intake per unit

weight gain of the average mouse of each group at the end of the five
week period is illustrated in Table VIII.

The castrate controls and

castrate animals treated with testosterone propionate appear to be the
most "efficient" groups for both food and water consumption.

These

groups require less food and water per gram of body weight gain.

As

found in experiment one, the castrate mice treated with both testos­
terone propionate and thyroprotein are the least "efficient", consuming
more food per unit gain in weight.

This substantiates the hypothesis

that the male hormone used has a synergistic action with thyroprotein.
The action is apparently on the process of metabolism and the two

TABLE VII
AVERAGE FOOD AND WATER CQNSOMPTION/MOUSE/DAT
(EXPERIMENT TWO)
2A

Week

2B

2C

Castrates Castrates
+
Castrate *■ Thyro­
Controls protein Pest. Prop.
(0.05$)
(10.2 mg.)
■-

2D

2E

Castrates
+ Thyro­
protein
Normal
(0.05$) + Controls
Test. Prop.
(10.2 mg.)

2F
Normals
+ Thyro­
protein
(0.05#)

Water Consumption (in Grams)

I

7.8

11.2

7.5

13.2

7.7

9.6

2

8.1

14.1

8.2

15.6

8.7

11.6

3

9.4

15.9

8.6

12.2

9.9

11.9

4

8.8

14.9

9.3

12.1

10.3

12.0

5

8.7

16.4

10.2

14.5

11.3

14.6

Aye.

8.5

14.5

8.8

13.5

9.6

11.9

Food Consumption (in Grams)
1

3.8

4.2

3.7

4.5

4.3

4.3

2

4-1

5.4

4.0

5.3

4.5

5.5

3

4.2

6.3

4.3

5.4

4.7

6.0

4

4.2

6.2

4.4

6.1

4.9

6.1

5

4.1

6.2

4.6

5.5

4.7

6.0

Ave.

4.1

5.6

4.2

5.3

4.6

5.6

41

TABLE VIII
AVERAGE POOD AND WATER-BODY WEIGHT GAIN RELATIONSHIPS PER MOUSE
(EXPERIMENT TWO)

Group

Description

Five
Weeks
Total
Food
in
Grams

EA

Castrate controls

143.4

299.6

10.3

13.9

29.1

SB

Castrates plus
thyroprotein
(0.05$)

199.0

508.5

12.5

15.9

40.7

148.2

306.7

10.6

14.0

28.9

187.3

473.9

8.2

22.8

57.7

SC

2D

Castrates plus
testosterone
propionate
(10.2 mg.)
Castrates plus
thyroprotein
(0.05$) plus
testosterone
propionate
(10.2 mg.)

Five
Weeks
Total
Water
in
Grams

Average
Five Wk.
Gain in
Body Wt.
in
Grams

Food
Water
Intake
Intake
Per Unit Per Unit
Wt. Gain Wt. Gain
in
in
Grams
Grams

2E

Normal controls

161.9

336.4

8.6

18.8

39.1

2F

Normals plus
thyroprotein
(0.05$)

195.9

418.2

9.3

21.2

45.0

42

substances together raise the rate of "utilization'* of energy above the
threshold of the anabolic effect.

An alternative hypothesis might be

that the two substances are acting together in reducing the absorption
of food and water from the digestive traot.

The singular similarity

of food and water Intake per unit weight gain for castrate controls and
for castrate animals treated with testosterone propionate shows the
lack of male hormone effect when unassociated with thyroprotein treat­
ment.

Also, the close correlation of water "efficiency" with food

"efficiency" for each group lends greater reliance to the data.

The

data also show the normal controls to be not as "efficient" in gaining
weight as are the castrate controls, the castrates receiving thyro­
protein, or the castrates treated with testosterone propionate.
In this second experiment, an attempt was again made to obtain
reliable data on body length increase assan adjunct to the body weight
increase in evaluating the growth effects of the hormones.

The fol­

lowing figure illustrates the linear relationships among the groups
(figure 7).

The millimeter increase in body length shows the thyropro-

tein-treated castrate mice to have a two-millimeter lead over the cas­
trate controls.

Inasmuch as the groups dropped off in body weight in

the fifth week of the experiment and inasmuch as the body length would
not be as flexible as the body weight, greater significance might lie
in the length measurements as a growth criterion.

The parallelism be­

tween length data of experiment one and experiment two is fairly close.
..Again more evidence of synergistic action of the two hormones arises
from the data.

Group

ZD

shows the same catabolic action of simultaneous

administration of the hormones, i.e., at these high dosage levels as is
shown in the food consumption pattern.

ooi

I

—

—

I

I

o u

ro

o

ro

oi

oi o

CASTRATE CONTROLS

ro

CASTRATES + T.R

ro

CASTRATES + T.PROR

ro
o

CASTRATES + T.P.
+ TPR0P.

ro
m

NORMAL CONTROLS

o °
;o
O

-fc

oi o

tn

u»

o

1------- 1------- 1------- 1-------1------- 1-------r~

ro>
CD

gj

figure 7

figure 7. Increase in Length of Mice in Experiment Two Over a five
Week Period. Thyroprotein Dosage at .05% Level of Peed and Testos­
terone Propionate Dosage as 10.2 mg. Pellet.

MILLIMETER INCREASE IN LENGTH (HEAD TO TAIL)
_

ro
*n NORMALS + T.P.
»

»

i i

1---- 1---- 1____ i____ i

•

44

The date on seminal vesicle-ventral prostate weight shows, like in
experiment one, that the dosage of testosterone propionate was still too
high; evidence indicated also by body weight data.

These results were

used to establish the male hormone dosage in the third experiment where
dilution of the pellet with cholesterol reduced the dosage by one-half.
The weights of the sex accessories are indicated in Table IX and pictorially in J'igure 8.

On the basis of equivalent body weight, the

average weight of the sex accessories of group
of group 2E.

SC

is greater than that

The addition of thyroprotein to the animals' diet results

in an increase in seminal vesicle-ventral prostate weights.

Caridrolt

and Arvy (1942) and Masson (1947) found the same effect of thyroprotein
on the sex accessory weights.

Experiment Three
The third experiment was established in somewhat the same manner
as the previous experiments but with the following differences:

The

first and main change was made in the testosterone propionate dosage.
Dr. Kbehakian while at the Steroid Hormone meetings in Madison, Wis.,
September, 1948, suggested the method he used successfully in reducing
the absorption rate of the male hormone.
by Shimkln and White in 1941.

This method was used previously

Cholesterol is mixed with the testosterone

propionate at various percentages and pellets are made from the mixture.
In the method used here, cholesterol (from the spinal cord of cattle)
was ground up finely in an agate mortar with an equal weight of the
male hormone to make a 50% mixture (Kbehakian, 1946a).
placed in an oven and brought up to 143° c.

This mixture was

In this melted state, the

mixture was taken up in a warmed pipette and released drop by drop into

45

TABLE IX
AVERAGE WEIGHTS OF SEX ACCESSORIES AT END OF FIVE WEEKS
(EXPERIMENT TWO)

Group

Description

Average
Body Weight

Average
Seminal VesicleVentral Prostate
Weight Per 25 Gto.
Body Weight

2A

Castrate Controls

28.9 gm.

Negligible

SB

Castrates plus
thyroprotein
(0,05$)

51.0 gm.

Negligible

28.8 gm.

202 mg.
±9*

26.7

SIS mg.
±17

SO

2D

Castrates plus
testosterone
propionate
(10.2 mg.)

Castrates plus
thyroprotein
(0.05$) plus
testosterone
propionate
(10.2 mg.)

2E

Normal controls

27.8

142 mg.
±11

2F

Normals plus
thyroprotein
(0.05$)

28.0

149 mg.
±9

* Standard error.

46

l-^
£
>-

oma

2A
2B
2C
2D
2E
2F

z
(9
■l
R
A
\

K
?

CASTRATE CONTROLS
CASTRATES + T.P. (.05%)
CASTRATES + T.PROR (I02MG.)
CASTRATES + T.PROP. (10.2MG.)+T.P. (.05%)
NORMAL CONTROLS
NORMALS + T.P. (.05%)

o MG.
£225
< 200|-

oc

2 I75F
UJ

> 1501cri
bJ 125
>
100
2

75

"
u
<

50h

UJ

25

figure 8
2R

2B

2C

2D

I I

2F

GROUP

Figure 8. Relationship of Sex Accessory Response to Various Treat­
ments of Testosterone Propionate and Thyroprotein,

47

distilled iee water.

As the drops struck the cold water, they solidi­

fied and dropped to the bottom.

They were left in the beaker and

placed in a refrigerator for 24 hours.

They were then removed and

dried in a desiccator to constant weight.
in a cool place until used.

The pellets were stored

The average weight of the pellets formed

in this manner was 10 mg, thus the pellet contained 5 mg. of testoster­
one propionate.

Pellets were also made with 75$5 cholesterol and 25$

testosterone propionate thus containing 2.5 mg. male hormone in a 10
mg. pellet.
A second change made in the third experiment was the use of indi­
vidual cages for the miee rather than keeping them in a large cage.

For

this purpose, a large battery of cages was purchased which housed 140
mice individually.

A photograph of the whole battery and a photograph

of an individual cage is shown below.
inches high, and 5 inches wide.

The cages were 7 inches long, 5

The sides and back of the cages were

solid metal; front and bottom were made of 1/4 inch galvanized wire
mesh.

The change from group caging to individualized caging was made

primarily to obtain statistically treatable data on food and water con­
sumption.

Its use resulted in certain other advantages however:
1.

No necessity for marking animals.

2.

Animals going off feed can be detected quickly and
eliminated from the records.

5.

Less aggressive animals have a chance at food and
water.

4.

Certain animals (food wasters) can be detected and
eliminated from the records.

49
5.

Spreading of disease is minimized and diseased
animals can be detected more easily,

Dr. Wolterink of the Department of Physiology and Pharmacology points
out however, that some workers say individualized cages offer some dis­
advantages over group caging in that there is supposed to be lessened
food and water consumption with individualized caging.
is supposed to stimulate better feeding.

Gregariousness

These observations are not

borne out by the results of the experiment reported here.

There is

actually a small, but statistically insignificant, increase in food con­
sumption.
Another change from the previous two experiments was made in the
addition of another group.

A group of 15 mice were treated with sub­

cutaneous Injections of testosterone propionate in oil three times week­
ly.

This was used as an added control for the pellet-treated group in­

asmuch as it is clearly established that subcutaneous injections are
absorbed at a very uniform rate (Bulbring and Burn, 1935).

A fourth

change from the earlier experiments was the reduction of the experi­
mental period from five weeks to four weeks.
The mice arrived from Rockland Farms at an average weight of about
16 grams and were grouped according to the scheme indicated in Table XL.
Groups 3B to 3F and 31 were castrated on June 8th by the same techniques
previously described.

Testosterone propionate-cholesterol pellets were

inserted into the nape of the neck of the animals shortly after castra­
tion.

Two days were allowed for operative recovery.

On June 11th,

the mice were distributed into their individual cages and placed on
their requisite food regime.

Food and water consumption for each mouse

was obtained every other day and averaged for daily values.

Body weight

so
records were made every fourth day rather than weekly.

This provided

more points to the growth curve and quicker indication of the effects
of various treatments.

Group I was injected thrice weekly with 0.47

mg. testosterone propionate in corn oil.

Kochakian showed in 1940,

that subcutaneous injections in mice given three times per week of
testosterone propionate in oil showed no different results from daily
injections.
After computing body weight gains on June 30th, it was noticed
that the animals treated with thyroprotein were beginning to lose weight
and so these animals were returned to normal Purina diet on July 1st.
Since chronic thyroidal stimulation does not stop immediately with the
cessation of treatment, but follows a characteristic decay curve ex­
tending over about four weeks (W. T. Salter, 1940), it was expected
that the effect of the early stimulation would carry over through the
fourth week.
again.

The next weighing period showed the weights going up

Group I were on normal diet for the first two weeks, then were

placed on Protamone for one week and returned to normal diet during the
fourth week.
At the end of the experimental period, the animals were weighed
and sacrificed.

The pellets were recovered in the usual manner.

Semi­

nal vesicle-ventral prostate glands were removed and weighed immediately.
The average gain in body weight of the animals in the different
groups at the end of four weeks is shown in Table X.

The same data trans­

lated into curves is illustrated in Figures 9 to 15.
Figure 9 shows the cumulative weight gain of the castrate controls
(3A) as compared to the thyroprotein-treated castrates (3B).

The curve

for the latter group is above that for the castrate controls, showing

TABLE X
AVERAGE CUMULATIVE WEIGHT GAINS (IN GRAMS) OF MICE IN
EXPERIMENT lirHRH!W
Experimental Plan
Group No.

Description

Gain Gain Gain Gain Gain Gain Gain
by
by
by
by
by
by
by
4th 8th 12th 16th 20th 24th 28th
Day Day
Day Day Day Day Day

3A

13

Castrate controls

2.9

4.3

6.5

8.3 10.3 11.2 12.3
±.8*

3B

11

Castrates plus
thyroprotein (0.05$)

3.4

6.2

8.1

9]6 10.3 12.4 13.1
±.9

3C

9

Castrates plus tes­
tosterone propionate
(5 mg.)

3.6

5.6

7.6

8.8

Castrates plus tes­
tosterone propionate
(2.5 mg.)

3.9

5.8

8.7 10.5 11.8 13.6 15.1
±.9

3.6

6.2

8.2

3.0

5.5

8.6 10.6 12.0 14.4 14.9
±.8

3D

3E

3F

11

11

7

Castrates plus
thyroprotein (0.05$)
plus testosterone
propionate (5 mg.)
Castrates plus
thyroprotein (0.05%)
plus testosterone
propionate (2.5 mg.)

9.6 10.7 10.8
±.8

9.3 10.1 11.7 12.1
±.6

3G

11

Normal controls

3.2

5.8

7.5

3H

10

Normals plus
thyroprotein (0.05%)

3.6

6.2

8.7 10.4 U . l 13.2 L3.1
±.5

31

12

Castrates plus
thyroprotein (0.05%)
third week plus tes­
tosterone propionate
(0.47 mg. in oil

3.7

5.9

7.7

3/week)

♦Standard error.

8.5

9.7 10.6 11.2
±.5

9.1 10.4 12.2 L3.0
±.6

52
the anabolic effect of thyroprotein on the animals.- On the 20th day,
the carve begins to level off indicating the disappearance of the ana­
bolic action.

This same condition was found in every group on thyro­

protein at this time while it was not found in the other groups (see
Figure 11).

The natural assumption to be made was that the level of

thyroprotein was still too high.

The thyroprotein feed was immediately

replaced with normal feed and the next ten days showed a spurt in the
growth curves of these groups.

The remarkable similarity of the change

in growth curves at the 20th day is noted in Figure 11.

Except for the

slight dip in the growth curve of group 3B during the 16th to 20th days,
as seen in Figure 9, the curves of both groups, i.e., 3A and 3B would
be parallel with about a 1 gram advantage to the group on thyroprotein.
Figure 10 shows the effects of two different dosage levels of tes­
tosterone propionate on the growth of mice.

For the first two weeks

both groups on testosterone propionate are above the castrate control
animals.

At about 14 days, the group on the 5 mg. pellet does not show

as great a gain in body weight as does the castrate control group.
Again this Indicates that the dosage here is too high and after an ini­
tial accumulation begins to have a catabolic effect.

Growth of the cas­

trates receiving 2.5 mg. testosterone propionate is rapid with steady
climb and at the end of the experimental period each mouse averaged al­
most three gm. more in weight gain than the castrate control animals.
The same pattern is shown in Figure 11, where the groups were treated
simultaneously with thyroprotein and male hormone.

The addition of the

thyroprotein raised the level of the three curves; compare, with cas­
trate controls and castrates plus testosterone propionate alone.

53

i

!
3A CASTRATE CONTROLS
3C CASTRATES ♦ T.PRORBMG.)
3 0 CASTRATES + T. PROP (2 3 MG)

3A CASTRATE CONTROLS
3B CASTRATE+ T.RIC6X)

s'

=6

figurt 9

14’

3 8 CASTRATES* T.R (05%)
3E CASTRATES + T.R (05%) + T.PROP.
(5MG.)
3F CASTRATES* T.R(OSS)
*T P R 0 R fc 5 M G )
/

flgura II
DAYS

flguralO

3G NORMAL CONTROLS
3H NORMALS* T.R.(.06%)

figurt 12

THYKOmorDW
DAYS

Figures 9, 10, 11 and 12. Growth Curves of Mice in Experiment Three
Over a Twenty-Eight Day Period.

54

AVE. CUMULATIVE

WEIGHT

GAIN

<314

3C CASTRATES ♦ T.PROP (5MG)
3E CASTRATES* T.PROP.(5MG)
+ T.P. (.05%)

3A NORMAL CASTRATES
31 CASTRATES*T.PROP INJECTIONS
* T.R (.05%)

figure 13

figure 14
24
DAYS

DAYS

3D CASTRATES * T.PROP. (2.5 MG.)
3F CASTRATES *TPROP.
£5MG)*T.R(D5%)
/'y

o4

DAYS

i

Figures 13, 14 and 15. Growth Curves of Mice in Experiment Three
Over a Twenty-Eight Day Period.

55
Figure 14 shows the growth of group 3E (castrates plus testosterone
propionate 5 mg. plus thyroprotein) as compared to the growth of group
3C (castrates plus testosterone propionate 5 mg.).

Figure 9 was com­

pared previously and shows the same features as Figure 13.

Figure 15

indicates that the anabolic action of testosterone propionate may have
allowed little room for the action of the thyroprotein addition.

Zither

the physiological limit to the stimulation of the growth is reached or
the high dosage of thyroprotein is preventing an additive effect.

From

the tendency of group 3F*s growth curve to move slightly upwards when
the thyroprotein was withdrawn on day 20, apparently the latter hypo­
thesis was the correct one.
The analysis of the food consumption data shows that again groups
treated with thyroprotein are consuming more food and water than the
groups not so treated.

Table XI below indicates the relationships.

The

thyroprotein treatment Increases food consumption by about 1 1/4 gm.
dally while the water consumption is increased by about 1 1 / 2 gm.

The

animals treated with male hormone show no apparent difference in food
and water consumption from that of control animals; however,the food
consumption per unit weight gain is smaller.

See Table XII.

In each

case those groups treated with thyroprotein show a higher food con­
sumption/weight gain ratio than the comparable group.

Apparently the

combination of thyroprotein and testosterone propionate at the 2.5 mg.
level produces the greatest growth at low level food consumption.

The

group injected with testosterone propionate also shows : good weight gain
at relatively low food intake.

TABLE XI
AVERAGE DAILY CONSUMPTION OF FOOD AND WATER/MOUSE
(EXPERIMENT THREE)

Group

Food
Intake

Water
Intake

Food
Intake/Unit Wt.
Gain

5.5 gm.
±,

2*

8.5 gm.
±.5

151.4
■ 12.3 gm.
12.3

6.7 gm.
±.3

10.5 gm.
±.5

175.2
■ 13.3 gm.
13.1

Castrates plus tes­
tosterone propionate
(5 mg.)

5.1 gm.
±.3

8.0 gm.
±.3

154.2
- 13.9 gm.
10.8

Castrates plus tes­
tosterone propionate
(2.5 mg.)

6.0 gm.
±.2

7.5 gm.
±.4

156.5
- 10.4 gm.
15.1

Castrates plus tes­
tosterone propionate
(5 mg.) plus thyro­
protein (0.05$)

7.1 gm.
±.2

9.5 gm.
±.4

183.9
- 15.2 gm.
12.1

Castrates plus tes­
tosterone propionate
(2.5 mg.) plus thy­
roprotein (0.05$)

7.0 gm.
±.2

8.0 gm.
±.4

182.1
- 12.2 gm.
14.9

Description

3A

Castrate controls

3B

Castrates plus
thyroprotein (0.05$)

30

3D

3E

3F

3G

Normal controls

6.1 gm.
±.2

7.5 gm.
±.4

138f2
• 12,3 gm.
11.2

3H

Normals plus
thyroprotein (0.05$)

6.9 gm.
±.2

10.0 gm.
±.6

180.6
* 13.6 gm.
13.1

31

Castrates plus tes­
tosterone propionate
(0.47 mg. in oil
3/week) plus thyro­
protein (0.05$)
third week

5.8 gm.
±.1

7.5.0tt.
±.4

144.7
- 11.1 gm.
13.0

♦standard error.

97
Experiment Eour
The results of the experiment above give good evidence of the growth
effect of thyroprotein given as 0.05% of the feed and of the growth ef­
fect of the 2.5 mg. dosage of testosterone propionate.

The nature of

the experiment suggested the idea that perhaps the dosage levels of
both hormones could be lowered to the point where no response in growth
would result from the administration of either hoxmone alone but that
a synergistic action if present would show up upon simultaneous ad­
ministration of thyroprotein and testosterone propionate.

The idea was

also considered as to the possibility of obtaining a growth effect with
a dosage of the male hormone so small as to produce no androgenic ef­
fect.^* Experiment four was established to test the above hypotheses.
In this phase, the thyroprotein level was reduced to

0.025%

of the feed.

Testosterone propionate was administered at two different levels:

one

at 2.5 mg. in an attempt to duplicate part of experiment three, and the
other at the 1.5 mg. level to test the hypothesis above.
The experimental plan for experiment four was as follows:
Group

Description

4A

Castrate controls

4B

Castrates plus thyroprotein (0.025$)

40

Castrates plus testosterone prop.

(2.5mg.)

4D

Castrates plus testosterone prop.

(1.5mg.)

George W. Thorn of the Department of Medicine at Harvard asked at one
of the Conferences on Metabolic Aspects of Steroid Compounds, if clini­
cally useful protein anabolism could be induoed by amounts of testos­
terone propionate Insufficient to induce masculinization.

58
Group

Description

4E

40

Castrates plus testosterone
plus thyroprotein
Castrates plus testosterone
plus thyroprotein
Normal controls

4H

Normal controls plus thyroprotein (0.025%)

4F

prop. (2.5 mg.)
(0.025%)
prop. (1.5 mg.)
(0.025%)

One hundred and forty male albino mice were received from Rockland
Barms at an average weight of 11 gm.

One hundred nineteen mice were

castrated and the incisions closed by surgical clips rather than with
silk sutures as was done in previous experiments.

The animals were

treated aseptically during the operation and dusted with sulfa powder^
after the incision was closed.

Immediately following the castrations,

the animals were placed in the individual cages provided for them.

Af­

ter a lapse of one day, 60 mice had testosterone proplonate-cholesterol
pellets implanted into the nape of the neck.

Thirty of these mice re­

ceived a single 10 mg. pellet containing 2.5 mg. testosterone propionate
and the other 30 received a single pellet containing 1.5 mg. of testos­
terone propionate.

The remainder of the pellet in each case was made

up of cholesterol.

The operated animals were allowed one day for opera­

tive recovery and were then weighed and placed on the desired food and
water regime.

The battery of individual cages and the laboratory con­

ditions under which the animals were placed was the same as in the pre­
vious experiment except that there was question as to whether the airconditioning system would maintain temperature under conditions of sum­
mer weather outside. Dally temperature records of the animal laboratory
revealed a variation no greater than 2 degrees on either side of the
mean temperature which was 74°.
^Containing urea, sulfanilamide and sulfathiozole.

59
The animals were weighed every 4th day and a record of food and
water consumption was taken every other day for the length of the ex­
periment (4 weeks).

At the end of the experimental period, the ani­

mals were weighed and sacrificed.

The stomach and intestines were re­

moved, washed clean with distilled water and replaced into the abdomen.
The animals1 seminal vesicles and ventral prostate glands were dis­
sected out, weighed and returned to the animal.

Testosterone propionate

pellets were recovered and treated as described previously.

Bata from

the earlier experiments indicated that some of the pellets weighed
more at the end of the experimental period than at the start.

This

was again verified here and search of the literature revealed the
phenomenon as one not uncommon to pellet usage.

Folley (1942 and

1943) reports the formation of "ghost" materials around implanted
tablets of synthetic oestrogens.

However, from the size of sex ac­

cessories, no apparent Inhibition resulted here from these scleroproteln "ghosts".
After cleaning the digestive tract, weighing sex accessories and
removing pellets, the carcass1 was weighed and recorded as carcass
weight.

Each animal was placed in a tared, labelled specimen bottle

and placed in the deep freeze (minus 5 degrees C.) for a period of one
week.

The bottles were then transferred to a drying oven (90° C.) for

another week.

This period was found satisfactory in a pilot study

carried out previously.
constant weight.

At the end of this time the animals were at

The animals were chopped crudely in the dried state

^The term carcass as used here includes the total animal body minus
contents of the digestive tract in contrast to the term as used by
some authors to mean the animal body minus internal organs.

60
in a Waring blender for one-half minute.
in a dry oven at 60° C.

They were stored in this form

These total mice were analyzed later for fat,

protein and ash in Dr. F. L. Wynd's Laboratory of Plant Physiology.
Analysis of the growth data reveals that the earlier hypothesis con­
cerning the synergistic action of the two hormones in question is still
tenable although statistical treatment shows the differences as not sig­
nificant.

The direction of the data, however, is so similar among the

various comparisons that it cannot be ruled out.

The growth curves of

the various groups are compared among one another in Figures 16 to 23.
In comparing the growth curves of group 4A and 4B of Figure 16, it
is seen that the curves are almost superimposed upon one another indi­
cating that the level of thyroprotein used gave little if any additional
s.

growth response to oastrated mice.

Figure 17 reveals that the levels

of testosterone propionate also caused no growth stimulation above that
of the castrate controls.

The growth curve of the group treated with

2.5 mg. testosterone propionate is contrary to the results of the same
dosage used on slightly heavier animals in experiment three where good
growth response was obtained.

Except for the weight difference (start­

ing weight for experiment three was on average of 16 gms. compared to
starting weight of 11 gms. in experiment four) there is no other clue
as to the reason for the discrepancy.

The curves of Figure 18 are al­

most identical in shape to those in Figure 17 except that they are high­
er, indicating that thyroprotein has boosted each of the groups somewhat
above its paired group, i.e., 4E above 40, and 4F above 4D.

The mice

treated with both hormones simultaneously show an increased growth rate
above the castrate controls; a phenomenon not evident when each hormone
is U39d alone.

This slight anabolic action although statistically

61

4A CASTRATE CONTROLS
4 C CASTRATES + TPROfi (25MG)
4 0 CASTRATES t T.PROP. (I.5M S)

4A CASTRATE CONTROLS
4 8 CASTRATES t T.R (.025%)

>8

u i4

figure 16

4 8 CASTRATES+ T. P. (.025%)
4 E CASTRATES + T.P.(.02S%)+T.PROP
.
(2 5 MS)
4 F CASTRATES+ T.R(.025%)
-4E
F T.PROP. (ISMS)

figure 17

4A CASTRATE CONTROLS
4 E CASTRATES t T.R (025% )
+ T.PROP. (25MG)

e0

figure B
24
DAYS

DAYS

Figures 16, 17, 18 and 19. Growth Curves of Mice in Experiment Four
Over a Twenty-Eight Day Period.

4A CASTRATE CONTROLS
4 F C A STR A T ES*T.PR O P(1.5MG)
*T.P.(.025% )

40 CASTRATES ♦ T. PR0RE5MG.)
4E CASTRATES ♦ T.PR0P.B5MG)
e T.R (.02910
(AH

AVE. CUMULATIVE

WEIGHT

GAIN

(GMS)

62

figure 20

4D CASTRATES ♦ T.PROP. (I.5MG)
4 F CASTRATES »• T.PROP. (1.5 MG.)

figure 21

4G NORMAL CONTROLS
4H NORMALS * T.P.(.025%)

AVE. CUMULATIVE

WEIGHT

GAIN

(GMS)

♦ T.R (.025%)

figure 23

20
DAYS

DAYS

Figures 20, 21, 22 and 23. Growth Curves of Mice in Experiment Four
Over a Twenty-Eight Day Period.

6as
insignificant is shown in both Figures 19 and 20 where in each case the
treated groups are above the untreated castrate controls.

Again the

duplicated parallels evince a "feeling" that the results show something
in spite of the cold calculating facts of statistics.
Groups 4G and 4H in Figure 23 again show that the level of thyro­
protein used had no great growth effect.

There is a slight increase

in favor of the thyroprotein-treated group.
As in the previous experiments, the sex accessories are used as
criteria for effectiveness or the "take" of the male hormone pellet.
The sex accessory weights indicated that the pellets containing 2.5
mg. testosterone propionate were having 71.0% of the action of normal
testicular secretion assuming that group 4G exhibited normal compara­
tive values for accessory gland weights.

See Table XII below.

The

groups treated with pellets containing 1.5 mg. testosterone propionate
showed seminal vesicle-ventral prostate effect of 51.0% of that of full
normal testicular response (group 4G).

The thyroprotein showed a slight

effect on uncastrated animals but no effect on castrated forms.
Individual records of food and water consumption reveal further in­
formation.

Although the growth effects of thyroprotein treatment was

negligible, the animals showed an increase in both food and water con­
sumption although not to the same extent as at the higher dosage levels.
The data of experiment three show

that thyroprotein treatment at the

level of 0.05% of the feed had an effect on appetite so as to increase
food consumption by 1.2 grams over that of the controls in the castrate
groups while the uncastrated animals on thyroprotein (0,05%) showed 0.8
gm. more food consumed daily than the normal controls.

In experiment

four there is an increased daily food consumption of 1.0 gm. in the

64

TAELS XII
AVERAGE WEIGHTS OF SEX ACCESSORIES AT END OF FOUR WEEKS
(EXPERIMENT FOUR)

Group

Description

Average Weight
Seminal VesicleVentral Prostate/
25 Gta. Body
Weight

Ratio:
Experimental Wts.
Control Wts.

4A

Castrate controls

Negligible

—

4B

Castrates plus
thyroprotein
(0.025%)

Negligible

—

4C

4D

4E

4F

Castrates plus tes­
tosterone propionate
(2.5 mg.)

127.5 mg.
±19.2*

0.78

Castrates plus tes­
tosterone propionate
(1.5 mg.)

85.4 mg.
, ±8.8

0.52

Castrates plus tes­
tosterone propionate
(2.5 mg.) plus thy­
roprotein (0.025%)

108.5 mg.
±18.3

0.66

Castrates plus tes­
tosterone propionate
(1.5 mg.) plus thy­
roprotein (0.025%)

84.5 mg.
±8.4

0.51

163.3 mg.
±15.0

1.00

181.8 mg.
±12; 5

1.11

40

Normal controls

4H

Normals plus
thyroprotein
(0.025%)
^Standard error. -

.

*5

025$)

thyroprotein-treated (0.

castrates over that of the castrate con­

trols and 0.6 gm. increase in daily food consumption of uncastrated ani­
mals fed thyroprotein (0.025$) over that of the normal controls.

The

consumption of mater followed the same pattern.
The evidence shows an apparent lag in growth following thyroprotein
treatment.

Low doses in the range used in this experiment result in in­

creased food and water consumption with no noticeable anabolic action.
Slightly higher doses show anabolic effects with increased food and
water consumption.

Still higher doses show excessively high food and

water intake with catabolic effects on the body.

Presumably the thyro­

protein is acting on two functions, one on the appetite, and the other
on tissue metabolism, and it appears that these two actions are not
directly dependent on each other.

If the food and water intake were a

straight line function of metabolism, then at middle range dosages the
food and water should be adequate to the metabolic needs of the body
rather than excessive so that the total effect is an anabolic one, and
at the higher range dosages the food and water intake on ad libitum
diet should be adequate instead of being insufficient so that the total
effect is a catabolic one.

Apparently then, there is a greater effect

on the appetite in the middle dosage range than there is either in the
lower or higher dosage range so that more food is consumed than is uti­
lized resulting in a net profit.

At the higher range, a large amount

of food is taken in but the high metabolic rate causes utilization of
all ingested food plus animal body food stores resulting in a net loss.
The actual average daily food and water intake is tabulated in the
table below (Table XIII).

The high levels of food consumption following

high doses of thyroprotein evidently do not contain sufficiently large

TABLE XIII
AVERAGE DAILY FOOD AND WATER CONSUMPTION/MOTJSE
(EXPERIMENT FOUR)
Group

Description

Food
Intake

Water
Intake

5.4 gm.
±.2*

6.5 gm.
±.3

4A

Castrate oontrols

4B

Castrates plus
thyroprotein
(0.025$)

6.4 gm.
±.2

8.7 gm.
±.6

Castrates plus tes­
tosterone propionate
(2.5 mg.)

4.9 gm.
±.2

5.6 gm.
±.3

Castrates plus tes­
tosterone propionate
(1.5 mg.)

5.0 gm.
±.3

6.2 gm.
±.4

Castrates plus tes­
tosterone propionate
(2.5 mg.) plus thy­
roprotein (0.025$)

6.2 gm.
±.3

7.8 gm.
±.4

Castrates plus tes­
tosterone propionate
(1.5 mg.) plus thy­
roprotein (0.025$)

6.5 gm.
±.4

8.0 gm.
±.8

40

4D

4E

4F

40

Normal controls

6.0 gm.
±.3

7.0 gm.
±.4

4H

Normals plus thyro­
protein (0.025$)

6.6 gm.
±.2

9.6 gm.
±.5

*Standard error.

67

quantities of the vitamins necessary to certain metabolic activities in
nutrition.

Drill (1943) reports increased need for vitamins A, B complex,

C and probably D with hyperthyroidism.

Another explanation for the net

loss incurred with high dosage of thyroprotein might be that the ab­
sorptive process in the digestive tract cannot keep pace with either
the food intake or the energy requirements.
In the search for a synergistic anabolic growth effeot, it became
apparent that any gross anabolic action noted would prove valuable only
if the quality of the gain in weight was known.

With this end in mind,

analysis of some 82 mice ranging from 7 to 14 in a group was made for
protein, fat, water content, ash and carbohydrate (by difference).

Par­

ticular advantage was obtained here inasmuch as the analyzed mice were
all approximately the same weight, i>e., at the end of the 28 day
growth period studied, no great gains in growth occurred in any one
group.
The dehydrated, crudely chopped animals were taken from their stor­
age place and analyzed first for total "fat" (i.e., anything in animal
carcass soluble in ether would come out as "fat"), A battery of 12 Gold­
fish Tat Extractors greatly facilitated the whole procedure.

By this

method, ether drips continuously through the mouse tissue for a four hour
period.

A total mouse of known weight in the crudely chopped state was

placed in a tall, thin, porous thimble of ceramic.

This thimble is

placed in the Goldfish Apparatus and is enclosed by a tared Goldfish
beaker containing 30 ml. of ether.

The beaker is sealed tightly into

the system by means of cork gaskets and lock rings.

Individual elec­

tric heaters evaporate the ether which rises in the apparatus, con­
denses, drops down into the thimble and seeps through the contents.

68
All ether-soluble substances are dissolved out and drip back into the
Goldfish beaker.

The cyclic evaporation and condensation continues in

this way for 4 hours.

At the end of this time, the thimble and con­

tents were removed and a glass thimble substituted for the ceramic one.
Continued evaporation of the ether and condensation traps it in the
glass thimble and most of the ether is thus freed from the contents of
the beaker.

The beakers were removed and warmed gently to remove the

last traces of ether and then placed in a drying oven at 100° C. for
30 minutes and transferred to a desiccator for 12 hours.

The beakers

and contents were weighed to the closest 10 thousandth of a gram.

The

fat-extracted tissue was put into large vials and stored in a dry oven.
The average amount of total "fat" in the various groups is expressed as
percentages of fresh and dry carcass and can be seen in Tables fry and
XXXI respectively.

The amount of total "fat" in each animal is shown

in Tables XIV to XXIX.
Upon completion of the fat extraction, the remaining carcass was
in a less gummy form and was readily ground into a fine powder in a
powerful Wiley mill.

The resulting powdered carcass was mixed thor­

oughly and returned to individual vials for further analysis.
The protein analysis was made by characteristic determination of
total nitrogen present as -NH^, -NH, and -NO (Triebold, 1946; Bradstreet,
1940).

The analysis also Includes of course ammonium compounds, amino

acids, urea and the more complex breakdown products of protein meta­
bolism.

A modified macro-Kjeldahl method was used as follows.

The

first phase of wet oxidation was carried out on a 0.15 gm. sample from
each powdered carcass weighed and wrapped in Wattman #1, size 9 filter
paper.

This sample was placed in a large Kjeldahl flask to which 25 ml.

TABLE H V
BODY COMPOSITION OF CASTRATE MICE EXPRESSED AS
PERCENT OF DRY WEIGHT (GROUP 4A)
No.

Proteins

Fats

Carbohydrates

Ash

1

69.23

16.28

0.69

13.80

2

68.81

12.51

4.24

14.44

3

58.14

20.26

8.54

13.06

4

70.81

12.92

2.85

13.42

7

59.69

26.45

2.68

11.18

9

67.88

14.69

3.44

13.99

10

62.75

21.78

4.32

11.15

li

62.63

22.03

3.20

12.14

12

72.44

10.09

3.28

14.19

13

64.44

19.23

4.17

12.16

14

58,50

25.99

4.12

11.39

Are.

65.03

18.38

3.78

12.81

70

TABLE XV
BODY COMPOSITION 07 THYROFROTEIN-TREA.TED (.025#) CASTRATE MICE
EXPRESSED AS PERCENT 07 DRY WEI GET (GROUP 4B)
No.

Proteins

Eats

Carbohydrates

Ash

1

63.63

22.40

1.92

12.05

8

71.50

11.58

3.11

13.81

4

72.50

11.26

2.28

13.96

5

62,63

16.01

8.43

12.93

7

64.81

20.54

2.31

12.34

10

71.56

13.39

2.45

12.60

11

67.63

14.28

4,04

14.05

12

68.25

12.54

4.84

14.37

13

63.81

19.98

4.19

12.02

14

70.63

13.00

1.42

14.95

Aye.

67.70

15.50

3.49

13.31

71

TABLE XVI
BODY COMPOSITION OF TESTOSTERONE PROPIONATE-TREATED (2.5 MG,)
CASTRATE MICE EXPRESSED AS PERCENT OF DRY WEIGHT (GROUP 4C)
No.

Proteins

Fats

Carbohydrates

Ash

2

66.38

19.61

1.44

12.57

3

72.13

15.09

---

13.04

6

69.44

14.34

2.48

13.74

8

57.50

26.66

3.71

12.13

12

60.13

29.74

---

10.99

14

64.63

19.17

3.94

12.26

15

66.13

20.56

0.58

12.73

Are.

65.19

20.74

1,58

12.49

TABLE XVII
BODY COMPOSITION OF TESTOSTERONE PROPIONATE-TREATED (1.5 MO.)
CASTRATE MICE EXPRESSED AS PERCENT OF DRY WEIGHT (GROUP 4D)
No.

Proteins

Fats

Carbohydrates

Ash

1

71.13

15.93

1.29

11.65

4

54.88

29.88

4.03

11.21

5

67.69

15.79

4.05

12.47

6

66.44

19.62

2.87

11.07

7

69.56

12.09

6.43

11.92

8

73.38

6.52

8.04

12.06

9

70.56

10,91

6.44

12.09

13

71.44

11.79

3.92

12.85

14

65.25

17.09

4.97

12.69

Aye.

67.81

15.51

4.68

12.00

73

TABLE m i l
BODY COMPOSITION OF CASTRATE MICE TREATED WITH TESTOSTERONE
PROPIONATE (2.5 MG.) AND THYROPROTEIN (.025$) EXPRESSED AS
PERCENT OF DRY WEIGHT (GROUP 4E)
No.

Proteins

Fats

Carbohydrates

Ash

1

76.94

8.69

---

15.42

2

65.69

17.13

3.43

13.75

3

59.94

28.68

1.99

9.39

4

73.50

11.27

2.32

12.91

5

69.56

10.01

4.21

16.22

6

48.94

35.73

6.16

9.17

7

68.6r3

15.33

3.50

12.54

8

69.06

12.26

3.90

14.78

10

70.06

13.25

3.22

13.47

13

60.00

26.26

4.45

9.29

14

68.69

16.94

3.39

10.98

15

71.56

12.12

3.08

13.24

Are.

66.88

17.31

3.26

12.59

TABLE XEX
BODY COMPOSITION OF CASTRATE MICE TREATED WITH TESTOSTERONE
PROPIONATE (1.5 MG.) AND THYROPROTEIN (.025$) EXPRESSED AS
PERCENT OF DRY WEIGHT (GROUP 4G)
No.

Proteins

Ffe.ts

Carbohydrates

Ash

1

71.19

8.20

5.71

14.90

2

73.56

12.37

1.84

12.23

3

73.44

6.37

3.98

16.21

8

71.25

11.01

4.04

13.70

9

74.44

11.75

1.72

12.09

10

72.50

8.39

5.93

13.18

12

58.13

30.50

1.13

10.24

13

59.63

24.97

4.45

10.95

14

63.75

19.37

4.50

12.38

Ave.

68.65

14.77

3.70

12.88

TABLE XX
BODY COMPOSITION 0? NORMAL MICE EXPRESSED AS
PERCENT OP DRY WEIGHT (GROUP 4G)
No.

Proteins

Pats

Carbohydrates

Ash

1

69.50

18.84

3.50

14.76

3

67.69

13.98

6.78

11.61

3

65.49

81.94

---

13.08

4

78.50

8.93

4.75

13.88

5

68.63

84.08

3.36

9.93

6

54.81

89.80

5.16

10.83

8

78.88

17.67

9

68.31

84.35

8.96

10.38

10

73.13

8.03

6.05

18.79

11

59.88

88.84

6.88

11.50

13

61.69

81.44

6.49

10.38

14

59.00

88.34

1.78

10.94

15

69.81

13.58

4.57

18.10

16

60.63

85.38

8.50

11.49

Ave.

65.14

19.48

3.35

18.09

16.87

76

TABLE X U
BODY COMPOSITION 07 THYROPROTEIN-TKEATED (.025$) MICE
EXPRESSED AS PERCENT OF DRY WEIGHT (GROUP 4H)
No.

Proteins

Fats

Carbohydrates

Ash

1

61.13

18.99

8.43

11.45

2

67.88

13.76

5.00

13.36

3

63.13

18.05

6.41

12.41

5

65.63

16.22

4.88

13.27

6

69.25

13.95

4.14

12.66

7

68.63

9.41

7.59

14.37

9

72.38

5.73

8.31

13.58

10

73.44

6.02

5.79

14.75

15

62.88

21.81

3.20

12.11

16

69.31

13.44

3.56

13.69

Are.

67.37

13.74

5.72

13.17

TABLE XXII
BOUT COMPOSITION OF CASTRATE MICE EXPRESSED AS
PERCENT OF NET WEIGHT (GROUP 4A)
No.

Moisture

Proteins

Fats

Carbohydrates

Ash

1

74.67

17,54

4.12

0.17

3.50

2

73.33

18.35

3.34

1.13

3.85

3

69.51

17.73

6.16

2.62

3.98

4

72.60

19.40

3.54

0.78

3.68

7

67.19

19.58

8.69

0.87

3.67

9

74.78

17.12

3.70

0.87

3.53

10

73.31

16.75

5.81

1.15

2,98

11

71.10

18.10

6.37

0.92

3.51

12

73.33

19.32

2.69

0.88

3.78

13

72.12

17.97

5.36

1.16

3.39

14

68.62

18.36

8.16

1.27

3.59

Ave.

71.87

18.20

5.26

1.09

3.58

78

TABLE 2XIII
BODY COMPOSITION OP THYROPROTEIN-TREATED (.025%) CASTRATE MICE
EXPRESSED AS PERCENT OP WET WEIGHT (GROUP 4B)
No.

Moisture

Proteins

Pats

Carbohydrates

Ash

1

70.81

18.57

6.54

0.56

3.52

2

74.60

18.16

2.94

0.79

3.51

4

71.11

20.95

3.25

0.66

4.03

5

73.22

16.77

4.29

2.26

3.46

7

69.95

19.48

6.17

0.64

3.76

10

71.37

20.49

3.83

0.70

3.61

11

70.27

20.11

4.25

1.19

4.18

12

73.17

18.31

3.36

1.30

3.86

13

70.55

18.79

5.88

1.24

3.54

14

72.31

19.56

3.60

0.39

4.14

Ave.

71.74

19.12

4.41

0.97

3.76

78

TABLE XXIV
BODY COMPOSITION OP TESTOSTERONE PROPIONATE-TREATED (2.5 MG.)
CASTRATE MICE EXPRESSED AS PERCENT 07 NET WEIGHT (GROUP 4C)
No.

Moisture

Proteins

Pats

Carbohydrates

Ash

2

68.46

20.93

6.19

1.00

3.96

3

71.84

20.31

4.25

---

3.67

6

71.99

19.45

4.02

0.69

3.85

8

67.50

18.69

8.66

1.21

3.94

12

66.07

20.40

10.09

---

3.73

14

71.83

18.21

5.40

1-11

3.45

15

68.84

20.61

6.41

0.17

3.97

Ave.

69.50

19.80

6.43

0.48

3.79

80

TABLE XXV
BOUT COMPOSITION OF TESTOSTERONE PROPIONATE-TREATED (1,5 MG.)
CASTRATE MICE EXPRESSED AS PERCENT OF WET WEIGHT (GROUP 4D)
No.

Moisture

Proteins

Fats

Carbohydrates

Ash

1

71.91

19.98

4.48

0.36

3.27

4

67.08

18.07

9.84

1.32

3.69

5

71.50

19.29

4.50

1.16

3.55

6

70.24

19.77

5.84

0.86

3.29

7

73.10

18.71

3.25

1.73

3.21

8

73.20

19.66

1.75

2.16

3.23

9

74.08

18.29

2.83

1.67

3.13

13

71.81

20.14

3.32

1.11

3.62

14

71.56

18.56

4.86

1.41

3.61

Are.

71.61

19.16

4.52

1.31

3.40

TABLE XXVI
BODY C(IMPOSITION OF CASTRATE MICE TREATED WITH TESTOSTERONE
PROPIONATE (2.5 MG.) AND THYROPROTEIN (.025%) EXERESSED AS
PERCENT OF WET WEIGHT (GROUP 4E)
No.

Moisture

Proteins

Fats

Carbohydrates

Ash

1

70.31

22.84

2.58

---

4.58

2

71.49

18.73

4.88

0.98

3.92

3

70.30

17.80

8.52

0.62

2.76

4

74.35

18.85

2.89

0.60

3.31

5

72.90

18.85

2.71

1.14

4.40

6

65.00

17.13

12.51

2.15

3.21

7

71.99

18.24

4.29

1.96

3.52

8

72.51

18.98

3.37

1.08

4.06

10

72.21

19.47

3.68

0.90

3.74

13

68.80

18.80

8.19

1.30

2.91

14

71.56

19.54

4.82

0.96

3.12

15

72.16

19,92

3.37

0.86

3.69

Ave.

71.13

19.10

5.15

1.02

3.60

82

TABLE XXVII
BOUT COMPOSITION OF CASTRATE MICE TREATED WITH TESTOSTERONE
HiOPIONATE (1.5 MG. ) AND THYROPROTEIN (.025$) EXFRBSSTO AS
PERCENT OF WET WEIGHT (GROUP 4F)
No.

Moisture

Proteins

Eats

Carbohydrates

Ash

1

74.45

18.19

2.10

1.45

3.81

2

73.30

19.64

3.30

0.49

3.27

3

75.60

17.92

1.55

0.97

3.96

8

74.15

18.42

2.85

1.04

3.54

9

72.01

20.84

3.29

0,48

3.38

10

74.52

17.98

2.14

2.00

3.36

12

70.33

17.25

9.05

0.33

3.04

13

65.57

20.53

8.60

1.53

3.77

14

70.73

18.66

5.67

1.32

3.62

Ave.

72.30

18.82

4.28

1.07

3.53

83

TABLE XXrai
BOUT COMPOSITION OF NORMAL MICE EXPRESSED AS
PERCENT OF WET WEIGHT (GROUP 4G)
No.

Moisture

Proteins

Eats

Carbohydrates

Ash

1

71.05

20.12

3.54

1.02

4.27

2

72.22

18.92

3.87

1.76

3.23

3

67.49

21.27

7.13

---

4.26

4

74.05

18.93

2.32

1.12

3.58

5

62.63

23.40

9.00

1.26

3.71

6

73.44

14.56

7.91

1.37

2.72

8

72.64

19.93

4.83

---

4.45

9

69.05

19.28

7.54

0.92

3.21

10

73.71

19.23

2.11

1.59

3.36

11

69.50

18.25

6.78

1.94

3.53

13

70.00

18.42

6.43

2.04

3.11

14

67.66

19.00

9.17

0.63

3.54

15

72.73

19.05

3.69

1.23

3.30

16

71.06

17.55

7.34

0.72

3.33

Ave.

70.52

19.14

5.83

0.97

3.54

TABLE H U
BODY COMPOSITION OP THYROPROTEIN-TREATED (.025#) MICE
EXPRESSED AS PERCENT OP WET WEIGHT (GROUP 4H)
No.

Moisture

Proteins

Pats

Carbohydrates

Ash

1

70.99

17.82

5.51

2.36

3.32

2

72.15

19; 02

3.83

1.28

3.72

3

71.48

17.83

5.15

2.00

3.54

5

71.62

18.63

4.60

1.38

3.77

6

72.65

19.01

3.82

1.06

3.46

7

73.74

18,14

2.47

1.87

3.78

9

74.42

18.95

1.47

1.69

3.47

10

73.68

19.34

1.58

1.52

3.88

15

70.39

18.62

6.46

0.94

3.59

16

70.89

20.18

3.91

1.03

3.99

Are.

72.20

18.75

3.88

1.52

3.65

TABLE XXX
SUMMARY OF DATA ON BODY COMPOSITION OF TREATED AND CONTROL
GROUPS OF MICE EXPRESSED AS PERCENT OF WET WEIGHT

Group 4
4A
Castrate controls
4B
Castrates plus
thyroprotein
40
Castrates plus tes­
tosterone propionate
(2.5 mg.)
4D
Castrates plus tes­
tosterone propionate
(1.5 mg.)
4E
Castrates plus tes­
tosterone propionate
(2.5 mg.) plus
thyroprotein
4F
Castrates plus tes­
tosterone propionate
(1.5 mg.) plus
thyroprotein
4G
Normal controls
4H
Normal controls plus
thyroprotein
*Standard error.

No.
Ani­
mals

Moisture

Proteins

Fats

Carbo­
hydrates

11

71.87
±.74*

18.20
±.28

5.26
±.60

1.09
±.18

3.58
±.08

10

71.74
±.50

19.12
±.39

4.41
±.41

0.97
±.17

3.76
±.08

7

69.50
±.91

19.80
±.85

6.43
±.39

0.48
±.21

3.79
±.07

9

71.61
±. 67

19.16
±. 26

4.52
±.78

1.31
±.17

3.40
±. 07

12

71.13
±. 69

19.10
±.41

5.15
±1.00

1.02
±.18

3.60
±.17

9

72.30
±1.14

18.82
±.47

4.28
±1.07

1.07
±.21

3.53
±.11

14

70.52
±.61

19.14
±.51

5.83
±. 64

0.97
±.17

3.54
±.13

10

72.20
±.44

18.75
±.23

3.88
±.51

1.52
±.15

3.65
±.07

Ash

TABLE XXXI
SUMMARY QF DATA ON BODY COMPOSITION OF TREATED AND CONTROL
GROUPS OF MICE EXPRESSED AS PERCENT OF DRY WEIGHT

Group 4
4A
Castrate controls
4B
Castrates plus
thyroprotein
4C
Castrates plus tes­
tosterone propionate
(2.5 mg.)
4D
Castrates plus tes­
tosterone propionate
(1.5 mg.)

No.
Ani­ Moisture
mals

Proteins

Fats

Carbo­
hydrates

Ash

11

---

65.03
±1.53+

18.38
±1.66

3.78
±.57

12.81
±.35

10

---

67.70
±1.19

15.50
±1.28

3.49
±. 65

13.31
±.33

7

---

65.19
±1.91

20.74
±2.14

1.58
±.63

12.49
±.32

67.81
±1.12

15.51
±1.55

4.68
±.69

12.00
±.21

9

4E
Castrates plus tes­
tosterone propionate
(2.5 mg.) plus
tliyroprotein

12

---

66.88
±2.16

17.31
±2.44

3.22
±.43

12.59
±.69

4F
Castrates plus tes­
tosterone propionate
(1.5 mg.) plus
thyroprotein

9

---

68.65
±2.12

14.77
±2.78

3.70
±.64

12.88
±.62

14

65.14
±1.55

19.42
±1.88

3.35
±.62

12.09
±.50

10

67.37
±1.29

13.74
±1.70

5.72
±.60

13.17
±.31

4G
Normal controls
4H
Normal controls plus
thyroprotein
♦Standard error.

of Kjeldahl Digestion mixture for Total Nitrogen^- was added.

The con­

tents of the flask were well shaken to get good wetting and 10 gm. of
powdered potassium sulfate was added to increase the boiling point and
speed up the oxidation process.

Five gm. of powdered sodium thiosulfate

was next added and the flask gently warmed on the digestion apparatus.
Wien frothing ceased, full heat was applied to the electric burners.
The flasks were rotated regularly throughout the first 15 minutes and
occasionally during the next hour.

The heating and digestion was con­

tinued for a period of two hours at which time the contents appeared
as a clear solution.

Twelve KJeldahls were run at a time including one

blank as a reagent control.

The flasks were allowed to cool and were

removed to storage racks.
In the second phase, the ammonium sulfate formed in the process
above was decomposed with strong alkali and the ammonia distilled off
into boric acid.

In this phase, 200 ml. of distilled water was first

added to the cooled digested mixture and mixed completely.

After cool­

ing again, 50 ml. of concentrated sodium hydroxide (50%) was added
slowly beneath surface of the flask contents.

A piece of mossy sine

was added to reduce bumping and to react to form a zincate (sodium
zincate) and hydrogen which keeps the solution well stirred.

Approxi­

mately 150 ml. of the mixture was distilled into an IShrlenmeyer flask
containing 50 ml. of Kjeldahl Boric Acid (indicator added)2. This

l-Made up of CuSO. (15 gm.), cone. HgSO. (8 lbs.), Salicylic Acid (125
gm.), Water (25 ml.).
^Indicator consists of: 5 parts 0.1% Bromcresol Green in 95% ethanol,
1 part 0.1% Methyl Red in 95% ethanol. 10 ml. of indicator added to
each liter borie acid.

88
boric acid modification (Masker and Wagner, 1933) somewhat simplifies
the method inasmuch as it eliminates the use of standard base.

The

boric acid reacts with and holds the ammonia which is titrated against
a standard acid in the next phase.
In the third phase of protein determination, a simple titration
was made of the ammonia in the boric acid solution against 0.0714 N
HOI.

This normality is useful in that each milliliter of acid used

corresponds to one milligram of nitrogen and the burette reading auto­
matically gives the nitrogen equivalent.

A duplicate was run for each

mouse carcass and after subtracting the amount of nitrogen in the blank,
the two values were averaged, and this average value multiplied by 6,25
(the protein conversion factor for total mouse tissue).

The amount of

protein per "computed weight" of sample gave the percentage of the pro­
tein in the sample.

The "computed weight* in each case was the actual

sample weight corrected for the weight of the fat which was removed so
that the "computed weight" acts as a true sample weight (as if fat were
still in it).

The amount of protein in each mouse can be seen in Tables

XIV to XXIX, while the average protein in each group is shown in Tables
XXX and XZn,
The next step in the analysis was determination of ash content.
After review of the literature on ashing techniques a method was arbi­
trarily chosen based on certain of the precautions necessary to obtain
accurate and verifiable results.

The process of ashing tissue, being

a very complex one, lends itself to the establishment of certain arbi­
trary techniques which are maintained throughout the ashing procedure.
Ashing methods are numerous and the common aspects of these methods
were incorporated into the procedure used here.

In the main, A.O.A.C.

89

Recommendations are followed.

Previous workers have found the follow­

ing factors to he the critioal ones:
1)

Rate of incineration.

2)

Temperature of incineration.

3)

Incineration time.

4)

State of sample.

5)

Volume of sample.

Ashing was started in a cold muffle furnace.

A 1 gm. sample was

enclosed in ashless filter paper (average ash per circle was 0.000064
gm.) and put into a platinum crucible.

Twelve crucibles were placed

at one time in the furnace where the temperature was brought gradually
to about 200° C. and maintained at that temperature for one-half hour.
This first gradual ignition prevents mechanical loss of ash which oc­
curs with rapid incineration.
The temperature of incineration which is probably the most criti­
cal factor in ashing tissues was fixed at 650° C.

Very high tempera­

tures, i.e., in the vicinity of 800°, cause volatilization of certain
elements such as K, Na, Cl, and P.

It also causes fusion of the mineral

matter which then prevents oxidation of the interior of the fused mass.
The water of hydration is lost in certain compounds at one temperature
and in other compounds at another temperature.

Ashing temperature also

effects the decomposition of carbonates to oxides.

The following car­

bonates decompose at the designated temperatures:
UgCOg between 300-400 degrees C.^
CaCOrj between 600-650 degrees C. (as precipitate)
SgCOg between 700-800 degrees C.

^Triebold, 1946.

90
o
The ashing temperature was set at 650 . At this temperature some of
the carbonates were decomposed to the oxide form and addition of satura­
ted ammonium carbonate at the end of the incineration period restored
the oxides to the carbonate foxm.

At a lower ashing temperature, the

time of ashing was extended beyond practical laboratory handling.

The

temperature was maintained by an electronically controlled thermostat.
The total time of incineration was set at 4 hours including the
half hour ignition time; the tissue was reduced to a fine greyish white
powder in this time.

A shorter period of time resulted in incomplete

oxidation and a dark ash.
The state of the material to be ashed here was such as to make no
precautions necessary.

The finely ground sample includes fairly large

amounts of bone, hair, nails, etc. which allows for sufficient perme­
ability of the air and more uniform oxidation.

If sample is too compact,

sand is ground into the tissue so as to spread the material and provide
more porous ash.

This was not necessary here.

Also a large mass of

the sample prevents diffusion of air into the more central portions
and results in less oxidation and higher ash weights.

In the experi­

ment here, a one gram sample was large enough for easy determination
of ash weight and small enough to allow thorough oxidation.
After addition of saturated ammonium carbonate to the cooled ash,
the ammonia and water were evaporated off gently in a drying oven at
100° C. and the crucibles transferred immediately to an oven at 200° C.
for two hours.

At the end of this time, the crucibles were removed to

a desiccator for four hours and then weighed.

The percentage of ash

in each mouse is shown in Tables XIV to XXIX and the average ash weights
for each group is shown in Tables XXX and XXXI.

91

The above analyses account for water content, protein, fat and
ash; by standard practice, carbohydrates were determined by difference.
The data were seanned carefully for extremes and on this basis
one animal was eliminated from the calculations.

The basis for judg­

ing the extremes was the statistical basis for extreme variates where
any animal showing variance less than three standard deviations from
the mean was considered within the normal range of variability.

The

criterion of three standard deviations from the mean was used because
of the comparatively small number of animals in each group.
None of the differences between groups of protein, fat, water,
ash or carbohydrate content were statistically significant when ordi­
nary methods of statistics are used.

By finer statistical manipula­

tions, i.e., by methods of analysis of variance, the results were still
shown to be on the borderline of significance.

By this method, the *t*

values of protein, fat, and ash were 0.737, 1.21, and 0.92 respectively*.
However, even though the statistical analysis is highly reliable, one
may take the liberty of assuming that the correlation of ^directions"
makes interpretation of the data necessary.
In each pair of experiments, i.e., 4A versus 4B, 40 versus 4E, 4D
versus 4F, and 4G versus 4H, the addition of thyroprotein to the feed
has raised the nitrogen storage of the group (see Table XXXI).

In each

case, this increased protein percentage has been at the expense of fats.
An early study by Cameron and Carmicheal (1920) showed disappearance of
fats in rats fed thyroid.

McKay and Sherrill (1941) reported a large

decrease in fat in thyroid-treated animals on a high fat diet as compared

*See Appendix for statistical analysis.

92

to the controls on a high fat diet.
at the expense of carbohydrates.
is reduced from 4.68$ to 3.70%.

In one case, it may also have been

Compare 4D with 4F where carbohydrates
In every case, there is an increase in

ash content of those groups treated with thyroprotein over and above
their "paired" group, as indicated in the following table (Table XXXII).
Group 40 at the 2.5 mg. level of testosterone propionate did not
show protein anabolism or gain in weight over that of the castrate con­
trols.

Group 4B with 0.025% thyroprotein did show protein anabolism

with no gain in weight over that of the castrate controls.

This indi­

cates a shifting around of metabolites by thyroprotein but not by the
2.5 mg. dosage level of male hormone.

However, the 1.5 mg. level of

testosterone propionate does show a protein anabolic action in tissue
percentage at least but no gain in weight over the castrate controls.
This is in agreement with previous work that shows higher levels of
male hormone acting in a definitely catabolic or even at equilibrium
state.
The question can be justly asked at this point as to why in ex­
periment three, the level of 2.5 mg. testosterone propionate produced
an anabolic growth effect whereas in experiment four the same dosage
level showed no such activity.

The only known difference between the

experiments was that the animals in the last experiment were about 10
days younger (about 5 gm. less in weight).

The difference in effect

might be explained by this age difference in that the younger animals
may be more sensitive to the dosage used than the older animals.

Phar­

macologists find wide ranges of sensitivity with age variations and a
ten day difference among mice might be equivalent to perhaps six months
difference in children.

Other explanations are not attempted here.

93

TABLE XXXII
AVERAGE ASH CONTENT/MOUSE FOR VARIOUS
(SOUPS (EXPERIMENT POUR)
Group

Ash

(% Dry

Carcass)

4A
4B*

12.81
13.31

4C
4E*

12.49
12.59

4D
4F*

12.00
12.88

4G
4H*

12.09
13.17

*Thyroprotein-treated group.

94

The combinations of thyroprotein with testosterone propionate at
either level of dosage (groups 4B and 4F) and with the normal male hor­
mone secretion (group 4H) show in each case a reduction in amount of
fat in the animals.

The increase in protein of group 4B over that of

group 44 is 2.67 percentage units.

The increase of protein in the 2,5

mg. testosterone propionate group over that of castrate controls is 0.16.
On the basis of one of the earlier hypotheses the combination of the
two hormones ought to produce an additive effect (i.e., 2.67 plus 0.16
equals 2.83).
only 1.85.

The protein difference between group 4E and 44 is actually

The increase of protein in the 1.5 mg. testosterone propio­

nate group is 2.78 percentage units more than that of the castrate con­
trols.

In combination with thyroprotein one might expect a total change

of 5.45 percentage units (2.78 plus 2.67).
duces a 3.62 change.

Actually the combination pro­

Here is a synergistic effect, at least from one

pharmacodynamic point of view which defines a synergistic effect as one
which results when the total effect of two agents is greater than the
effect of each agent taken individually.

This is in contrast to another

pharmacological definition, one by Sollman (1948) where synergism is a
simple algebraic summation.

Webster's synergism has the total effect

greater than the sum of the two effects taken independently.

Sollman

says "if the action of two agents is really identical, their mixture
would be equivalent to increasing the concentration of either, and
would therefore follow the Weber-Fechner law of diminishing returns
and appear deficient".
Evidence from Klnsel, Hertz and Reifenstein (1944) apparently lends
credence to the hypothesis that both thyroprotein and testosterone pro­
pionate act on the same metabolic intermediary, where testosterone

95

propionate induced a strongly positive nitrogen balance and gain in
weight in thyrotoxic patients characterized by increased urinary nitro­
gen and creatine excretion and by decrease in body weight.

Interpreted

from another point of view, it is difficult to believe that the two
hormones are working on the same metabolic intermediary if in thyro­
toxic patients where the thyroid hormone is present in large quantity
and available to the metabolic intermediary, the addition of another
substance acting on that intermediary should act any differently than
more of the same substance.

The thyroid hormone affects so many of

the endocrine glands that a simple interpretation may not be warranted,
Thyroprotein stimulates the adrenal cortex to additional production
of steroid hormones which may have an additive or antagonistic effect
depending on the quantities involved.

Certainly sufficient evidence

exists as to adrenal stimulation by thyroidal substances (Wallach and
Reineke, 1949).

Kenyon, et al, (1943b) show no reason to adduce the

adrenal cortex as a necessary intermediary in the metabolic response
of men to testosterone propionate inasmuch as the concentration of
serum electrolytes was unaltered.
In comparing the castrate groups receiving testosterone propio­
nate at levels of 2.5 mg, and 1.5 mg. with the castrate controls, it
looks as if the 2.5 mg. level has no nitrogen retentive ability in
very young mice but apparently assists in converting carbohydrates to
fats, while the 1.5 mg, level has nitrogen retentive ability at the
expense of the pool of substrates which would have gone into the fats.
This latter effect was postulated by Kochakian some years back,

Kenyon

(1944) maintained glycosuria in human patients and was not able to

96

promote conversion of the carbohydrates to anything else with testos­
terone propionate treatment.
The discrepancies here, however, indicate needed Investigation as
to the differences between "fat" and "thin" castrates among dogs and
man because the same apparent discrepancies occur there.

DISCUSSION

The relationships of the thyroid and testes hormones to the growth
and metabolism complex are examined in this dissertation from four as­
pects:

growth, tissue analysis, enzymes, and metabolic rate.

In specific dosages, the hormones used in this particular study
were shown by previous workers to increase body weight, to stimulate
nitrogen retention, and to increase water content of the body.

As­

suming these facts to be correct and capable of verification, it is
of great interest to pursue the mechanism of the reaction.

If the

mechanism of action of one hormone is the same as that of another hor­
mone, then when maximum anabolic results are produced by a certain
level of each hormone acting alone, a combination of the two hormones
should produce no greater results than each one alone.

If no greater

result is produced, then it can be assumed as stated above that the
mechanisms are the same unless one interjects the plaguing thought
that there is a limit to "how far an organism can be pushed".

It is

no doubt true that there are certain physiological limits established
by the hereditary pattern of the species beyond which the organism can­
not be made to go.
If, however, the gross growth data do not reveal a synergistic
effect which would allow for different mechanisms of action, a careful
detailed analysis of the carcass under treatment with the different
hormone8 and combinations thereof, might reveal information of value
in understanding a little more of the complex Metabolism-Energy-Enzyme
system.

97

98

In understanding the process, it is necessary to soan the total
mechanism of energy supply.
mater is ingested.

First, a certain quantity X of food and

The food contains water, proteins, fats, carbo­

hydrates, vitamins and minerals.

Assuming normal digestion and absorp­

tion^, the food is broken down into the typical products of the major
constituents of the food and "taken into the body".

Let us also assume

here that a greater intake of food and mater (quantity Y) results in an
increase in body meight.

Norn, if the percentage of proteins, fats, car­

bohydrates, vitamins, minerals and mater in the carcass remains the same
as at the lomer body meight, then the metabolic picture has not changed.
Hbmever, if the percentage composition of animal carcass T has changed
from the picture in animal X, it can be assumed that
1) the quality of the absorption in animal Y has changed
so that at increased food intake, certain components
are more readily absorbed in relation to the other com­
ponents, or
2)

the digestive products in animal Y are absorbed at the
same rate as in animal X but the products are thrown into
the energy pool, i.e., the glycerols as alpha-glycerophosphate work their may into carbohydrate metabolism,
as do the carbohydrates themselves whioh all leads tomard protein synthesis, or

3) mater is absorbed in animal Y at a greater rate than in
animal X, resulting in a uniform decrease in the percentage

^The assumption may not be justified in view of the evidence that thyroid
hormone causes increased absorption of carbohydrates. (Althausen, 1940;
Althausen and Stockholm, 1938.)

99

of other components based on wet weight but in an over­
all increase in body weight.
Some speculation on locus of action of testosterone propionate in
inducing protein anabolism in patients with hyperthyroidism was made
by Albright (1943b) as follows:
"1.

2.

3.
4.

That testosterone propionate causes such a demand for amino
acids that none are available for glueoneogenesis regardless
of the partition of carbohydrates and fats in the metabolic
picture.
Testosterone propionate may produce some alterations in the
metabolism so that it is no longer necessary to have as high
a ratio of carbohydrates being burned to fat being burned.
Testosterone propionate may facilitate the combustion of fat.
Testosterone propionate may convert fat into carbohydrate.1*

A number of hypotheses concerning the mechanism were laid down as fol­
lows and examined in the light of the evidence available:
Hypothesis A - The gain in weight would be proportionate among
the major components of the animal body, i.e., there would be an
increase in absolute weights of protein, fat, carbohydrate, water
and minerals.
Hypothesis B - The gain in weight would be due to a greater pro­
tein anabolism and would show up on a percentage basis at the
expense of the other components.
Hypothesis C_- The gain in weight would be due to greater protein
anabolism, but at the expense of fats with no change in percentage
of the other components.
Hypothesis D - The gain in weight would be due simply to an in­
crease in amount of water and the dry carcass would reveal no
differences in the major components.
Hypothesis E - The gain in weight would be due to an increased
amount of fat at the expense of the other components.

100

Hypothesis ? - The gain In weight would be due to conversion of
fat and protein into common substrates with subsequent conversion
to carbohydrates.
Hypothesis G - The gain in weight would be due to both increased
protein and water at the expense of the other components.
Support for Hypothesis B comes from various sources.

Protein ana­

bolic effects of the male hormone have been shown by Kbchakian in in­
creased muscular mass in treated mice.

Verification of greater nitro­

gen retention is reported by Johnston (1941) with children and Terroine
and Babad (1939) with rats.
Kenyon and Khowlton (1944) have "attempted to secure some infor­
mation as to the role of muscle in the initial deposit of nitrogen in
man" after treatment with testosterone propionate of a patient with
progressive muscular dystrophy.

The nitrogen retention was distinct

but not spectacular and the experiment did not elucidate the site of
the new tissue deposit.

They state:

"We do not understand now why the organism dissipates so much
feed nitrogen during recovery from protein losses and is hence so
dependent upon greatly augmented appetite during convalescence.
1.

2.

3.

The need of the body for the energy derived from protein
for ordinary maintenance needs does not seem to be of
major importance.
The failure of ordinary food to meet the precise amino
acid, and if you wish, vitamin requirements of the par­
ticular state of the animal may well be of importance,
but neither the composition of food nor the manner of
ingress of protein fragments precludes intensification
of nitrogen retention when a suitable stimulus to pro­
tein anabolism is given.
It has not, however, been shown that such stimuli as
have been administered precisely meet the requirements
of the organism during protein depletion. Until this
is done, we cannot assume that the body has omitted
wise synthetic measures from its economy or that it
has been unable to use those it has.

101

"4.

It is possible that we have focussed too much attention
on nitrogen itself, and that the carbon chains derived
from the amino acids are of much more importance for ob­
scure reasons during recovery than are the intact amino
acids. Such a purpose has been sought for the mechanism
of nitrogen loss after fractures described by Cuthbertaon
and by Howard, et al. Such carbon chains may serve tis­
sues in the course of repair after fractures, and may
serve a similar purpose in recovery from protein losses,4*

The importance of vitamins mentioned in point two above is brought
out by Peters and Rossiter (1939) who find that administration of thy­
roid or thyroxine increases the requirement of vitamin B,
Drill (1943).

See also

The vitamin B requirements vary with the caloric out­

put, which is raised by thyroxine.

The loss of weight accompanying

high thyroid administration is lessened by adequate intake of vitamin B.
Kenyon, et al. (1943a) after a study of anorexia in man concluded
that when anorexia limits the food intake to amounts insufficient to
favor material storage of protein, then the androgen will induce such
storage.

Here there is more efficient usage of available proteins or

stimulus to conversion of other types of food to protein or perhaps
protein is shifted into storage form whereas under the conditions of
anorexia, it might have been utilized for energy.
The concept of a dynamic equilibrium implies the presence of a
large protein pool in tissues which contributes to the plasma protein
and the latter contributes in turn to the cells and tissues.

There

are evidently little or no reserves of free essential amino acids and
in a deficiency of any one, cannot obtain it from the protein stores
(Cannon, 1949).

Thus with the known anabolic effect of testosterone

propionate and the known appetite-stimulating effect of thyroproteln,
the combination ought to resolve itself into sufficient amino acid sup­
ply through increased food intake to promote substantial growth effects.

102

"Tat* eunuchs lend support to Hypothesis E where there is direct
evidence of increased fat storage heneath the skin, however, the "thin”
eunuchs show the opposite effect.

The well known action of thyroid

in "reducing tablets" on the fat stores of the body is also evidence.
Hypothesis G is supported by data of Kenyon, et al. (1940) who
postulate the gain in weight of man after treatment with testosterone
propionate to be due largely to water held in association with the re­
tained salts and protein.

Koger, Hurst and Turner (1942) determined

the composition of thyroxin-injected female mice and compared the data
of the analysis with control mice.

"The fresh carcasses contained a

greater percentage of nitrogen and water, and a lesser percentage of
fat than did their controls".
Composition of
Dry Carcass
Protein
Eat ?6
Ash 96
Water

%

A resume of their data follows:
28 Day
Thyroxine-treated

Controls
54.0
30.4
10.6
63.7

59.6
26.1
10.3_______
65.7

Since steroids from both the testes and adrenal cortex (Thorne
and Eisenberg, 1939) influence the growth process, it is pertinent to
more complete understanding of the problem to study the role of en­
zymes.

Corticosterone prevents hypoglycemia of Addison’s disease

either by facilitating the formation of sugar from protein or by in­
hibition of sugar utilization by the tissues (Talbot, et al. 1943).
Gluconeogenesis is also stimulated by thyroxine.

Alanine, glycerose

and pyruvic acid are transformed to glycogen slowly.

The three-carbon

keto acid fragments of amino acids are important in gluconeogenesis,
thus glycine and the three-carbon acids are converted quantitatively
while the other amino acids have parts convertible.

103

Inasmuch as the enzyme phosphatase functions in carbohydrate
metabolism in several places such as phosphocreatine to creatine con­
version, phosphoglycerate to pyruvic acid conversion, and adenosine
triphosphate change to adenylic acid, the variation
the enzyme is of importance.

of quantity of

Although the liver is of key importance

other organs have been examined also for changes in phosphatase.

In

an extensive study of the normal mouse kidney Kochakian (1947) reports
that:
"The normal mouse kidney shows alkaline phosphatase in the
cells of the proximal convoluted but not the distal tu­
bules. It is present at the turn of Henle's loop and in
the capsule about the glomerulus. In the castrated mouse
the cells of the kidney decrease in size with a resulting
greater concentration of the enzyme in the cells. After
testosterone treatment there is an apparent *washing out*
of the 'alkaline* phosphatase in the distal end of the
proximal convoluted tubules with a somewhat greater con­
centration in the portion nearest the glomerulus. This
shift in the concentration of the enzyme suggests that the
kidney is striving to recover substances (phosphates?) as
quickly as possible from the glomerular filtrate.**
Androgens also affect the secretion
gland.

ofacid phosphatase by the prostate

The prostate gland of children contains little of this enzyme

and administration of testosterone propionate results in a small in­
crease.

Much evidence is available

on serum alkaline phosphatase where

of the effects of the male hormone
an increase i3 noticed after treat­

ment (Kochakian and Vail, 1947j Humm, et al. 1948, Kochakian, et al.
1948; and Bucbwald and Hudson, 1944).
The thyroid hormone also affects the serum phosphatase.

In infants

and children suffering from hypothyroidism, the serum phosphatase was
found to be abnormally low and adequate thyroid therapy restored the
phosphatase to normal (Talbot, et al. 1941).

Wilkins and KLeischmann

(1946b) report that the thyroid influences conversion of creatine into

104

creatinine directly or through an effect upon the enzymes controlling
the creatine-phosphocreatine cycle.

Sure, et al. (1941) report marked

creatinuria with a reduction in preformed creatinine in hyperthyroid
rats.
Concerning arginase, Kochakian (1947) reports that the addition
of 0.5 mg. testosterone to samples of incubation mixture containing
homogenisate of kidney did not produce any significant change in ar­
ginase.

Kidney arginase increases with testosterone propionate treat­

ment (Kochakian, 1945).

Since arginase occurs in the proximal con­

voluted tubules, Kochakian assumes that the cells in this tissue are
stimulated to recover for protein fabrication more of those materials
that ordinarily would be excreted.

Kochakian in 1945 postulated that

arginase has a greater function than in the urea cycle in the liver.
S. Zdlbacher (1938) as a result of extensive studies postulated that
one of the functions of arginase may be to provide suitable nitrogen
forms for protein synthesis.

Borsook and Dubnoff, (1943) provide evi­

dence for one synthetic process, the formation of glycoeyamine, in the
kidney by the transfer of the amidine group of arginine to glycine as
follows:

HN-C
N HZ
HN-C
H-NH
NH

+

CHNHo

?h 2

I
0 - C-OH

0 - C-OH
0 - C-OH

Arginine

Glycine

Glycoeyamine

105

The glycoeyamine is then methylated in the liver to form creatine.

In

order to explain the above, one must assume that arginase promotes glycinolysis of arginine in the kidney instead of hydrolysis as occurs in
the liver.

This is entirely plausible.

DuVigneaud (1942) showed the

significance of labile methyl groups in the diet and their relation to
transmethylation.

Isotopic methyl groups made their appearance in the

creatinine of the urine and in the choline of the blood.
No increase in arginase in the liver was produced with treatment
of mice with testosterone propionate while the expected anabolic effect,
however, was in evidence.

This is in good agreement with the above pos­

tulations.
Finally, inasmuch as both the thyroid and testes hormones affect
the metabolism of the organism it is pertinent to question the relation­
ships of the mechanisms.

Sandiford, et al. (1941) find that the basal

metabolic rate of 5 eunuchoids and 1 eunuch was increased by 10 points
each by testosterone propionate treatment.

They point out, however,

that the action is not similar to that occurring via the thyroid as
the pulse rate remained the same.

McCullagh and Bossmiller (1941a) get

the same effect with methyl testosterone and they also remark as to the
difference of effect from that of the thyroid.

They say that the cli­

nical picture does not resemble hyperthyroidism even when the B.M.R.
is raised as high as 38$.

Jones, et al. (1941) from their results pos­

tulate the effect of the male hormone to be directly on the tissues or
intermediately through some other gland.

In man, the hypermetabolism

whenever it appeared following steroid hormone treatment differed from
that occurring as a result of hyperthyroidism.

It was not accompanied

by tachycardia, palpitation, hyperpiesis or increased sweating.

A

106

qualitative as well as quantitative effect on metabolism was suggested
by the fall in respiratory quotient of more than half the subjects.
In some cases the R.Q. dropped to 0.690 (Chambers, et al, 1942),
In general, the evidence presented above indicates definite dif­
ferences in the mechanism whereby an anabolic effect is produced by
testosterone propionate and by thyroprotein.

SUMMARY AND CONCLUSIONS

The growth-stimulating effect of thyroprotein is substantiated.
The dosage of thyroprotein having the greatest anabolic effect on
the immature albino male mice used in this experiment is .05$ of the
feed.

The growth-stimulating effect of testosterone propionate is

substantiated.

The dosage of testosterone propionate having the

greatest anabolic effect on the immature albino male mice is 2.5 mg.
in pellet form under the skin.

A slight synergistic effect on growth

although statistically not significant Is obtained when thyroprotein
(.025$) is administered simultaneously with testosterone propionate
at the 2,5 mg. level in pellet form under the skin.
Food and water consumption increases with increasing dosage of
thyroprotein.

No such effect is noticeable with testosterone propio­

nate treatment.
Analysis of the carcasses of eight groups of mice under various
experimental conditions indicates an increase in protein in the groups
treated with thyroprotein (.025$) which is apparently at the expense
of fats.

The testosterone propionate-treated group (1.5 mg.) shows

higher protein content than the controls.

The combination of testoster­

one propionate (1.5 mg.) and thyroprotein (.025$) 3hows a synergis­
tic protein anabolic action.

This is viewed in the light of the known

weight depressing effect of larger dosages of each of the hormones.
The data herein described lend weight to the hypothesis that these
hormones are acting on different mechanisms of protein and fat meta­
bolism.

107

108

The relationship of these hormones to the enzyme-substrate pic­
ture of carbohydrate, protein and fat metabolism is now being investi­
gated energetically by a number of workers and the nert few years should
reveal fundamental links in the chain of events of tissue metabolism be­
tween enzymes, substrates and hormones.

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APPENDIX
TABLE I
METHOD 07 ANALYSIS 07 VARIANCE
Group

Group
A

Group
B

Group
C

Group
D

X1

*1

X1

*1

X2

X2

X2

X2

*3

H

H

«

«

•#

•

*

«

•

•

«

•

•

•

*

%

4

?
|5

gA

Group
Total

* T
All items
*x2

* x 4 -- Xl2+Z22+X32.
n - Tot. No. Animals in Group

N - Tot. No. of Animals
m 2

C T - —
• • N
A ■ All^i|ems

B -/«A2
I
\ nA
M

M

_

t£

+ SB2 +
M

M

*

nB

m

Sc2

M

M

^otal Sum of Squares

+

•

•

•

«

•

•

•

»0

■

Y .
/

Between Group Sum of Squares
ANALYSIS 07 VARIANCE 07 MEASUREMENT
Source of Variation

Degrees
of
Freedom

Sq.

B -

fb

-W
fb

120

£
K

E“A-B«

*E

M |h“

Within Groups - Error

F^Snedecor
Table)

A «

Total
Between Groups

Sum
of
Squares

Sw

121
For Significance Comparison Between Any Two Groups:
-\| w

S-

OZ *

-*aTZT' -

A

**"/

% -

.
-»

jv ,

^
-

_

*****

" V C — ^

+

**
i

.
-»

(rr.

* *__
*
J-^ T

<T'^'

The *t* value was also computed on the basis of the following
equation:
t

M , - M i

where:
t ■ "t" value and is compared with nt" tables for significance
at 5# or 10$ probability.
M

m

mean

s * ungrouped standard deviation 9

-J t(v-M)‘
'
A/-/

N - number of animals.

The standard errors were computed on the basis of Bessel’s formula: