CENTRAL NERVOUS SYSTEM summer: as
pRoLAch SECRETION
AND
EFFECT OF ANTERIOR PITUITARY HORMONES ON
INDUCTION or NORMAL AND NEOPLASTIC .MAMMARY
GROWTH m THE RAT

Thesis for the Deg!» of Ph. D.
MICHIGAN STATE UNIVERSITY
Purnachandra ‘Kashavrao "i’aiwalker
1964

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ABSTRACT
CENTRAL NERVOUS SYSTEM INHIBITION OF
PROLACTIN SECRETION
AND
EFFECT OF ANTERIOR PITUITARY HORMONES ON
NORMAL AND NEOPLASTIC MAMMARY GROWTH

by Purnachandra Keshavrao Talwalker

l. The effects of chlorpromazine, a central nervous
system (CNS) depressant, on mammary growth and secretion
was investigated in the rat. Chlorpromazine stimulated
mammary lobulo—alveolar (L-A) growth, and initiated mammary
secretion in estrogen-primed rats or maintained mammary
secretion in post-partum rats after litter withdrawal. In
the estrogen-primed rat, initiation of mammary secretion
normally requires prolactin and ACTH. These results indicate
therefore, that chlorpromazine promotes secretion of both
prolactin and ACTH by the anterior pituitary (AP).

2. The ability of the rat hypothalamus to inhibit
prolactin secretion by AP in yi££g_was determined by a 2-hour
incubation of rat AP. Homogenates or acid extracts of rat
hypothalamus inhibited prolactin synthesis and release by AP,
whereas acid extracts of cerebral cortex had no effect,
indicating the presence of a prolactin inhibiting factor (PIF)
in rat hypothalamus. Acetylcholine, epinephrine, norepine-
phrine, serotonin, histamine, substance P, oxytocin, arginine

or lysine vaSOpressin and bradykinin had no effect on pituitary

Purnachandra Keshavrao Talwalker

prolactin release, indicating that PIF is different from any
of these substances normally present in the hypothalamus.
PIF in the rat hypothalamus was dialyzable, showing that it
is a small molecule and perhaps a peptide similar to other
neurohumoral releasing factors. Acid extracts of hypothalami
from cattle, sheep, and swine origin also showed PIF activity.

3. The ig_vigg_and i§;yi£rg_capacity of the rat
"mammotropic" pituitary tumor (Furth, MtT.F4) to secrete
prolactin was determined. The prolactin contents of MtT.Fu
and normal AP-tranSplants were very low as compared with
normal AP ifl.§l§2° Considerable amounts of prolactin activity
could be detected in the blood plasma of rats bearing MtT.Fu,
but not in the plasma of cycling, estrogen primed, pregnant
or lactating rats, or in rats bearing a single AP-tranSplant.
MtT.F4 released considerable amounts of prolactin into the
medium during 3 or 6 days of culture, although the amounts of
hormone released were small than by normal AP in culture. It
is concluded that the MtT.Fu, like the normal AP removed from
hypothalamic inhibition, synthesizes and releases prolactin
at a very rapid rate, thereby retaining very little prolactin
in the tissue.

4. MtT.Fu was successfully tranSplanted into Sprague-
Dawley rats from the inbred Fischer strain of rat. However,
tumor growth and percentage ”take" in the Sprague-Dawley

strain were lower than in the Fischer strain.

Purnachandra Keshavrao Talwalker

The tumor in the Sprague—Dawley rats apparently secreted
prolactin, STH and ACTH as it does in the Fischer rat, and
thus remained functionally similar to the original tumor.

5. A MtT.F4 tranSplant stimulated mammary L-A growth
in adreno-ovariectomized Fischer rats. Also, injections of
combination of prolactin and STH stimulated mammary L-A
growth in adreno—ovariectomized—hyp0physectomized Carworth
CFN rats. This demonstrates that the AP hormones, prolactin
and STH, can promote mammary L—A growth in the apparent
absence of ovarian and adrenal cortical hormones.

6. Mammary tumors were induced in ovariectomized
Sprague—Dawley rats, with limited treatment with estradiol
or prolactin and STH, following a single DMBA (7, lE-dimethyl-l,
2-benzanthracene) feeding. No mammary tumors developed in
untreated ovariectomized rats fed carcinogen. These results
indicate that prolactin and STH can produce mammary tumors in
carcinogen treated rats in the apparent absence of ovarian
hormones. They also indicate that the pituitary hormones,
prolactin and STH, are essential during the initiating phase
of mammary carcinogenesis in the rat.

7. Differences in the susceptibility of the mammary
gland to chemical carcinogens were investigated by using 4
different strains of rats and 3 different carcinogens. The
mammary glands of Sprague-Dawley strain were much more
susceptible to production of tumors (33%) than the other

strains studied (O—7%, Carworth CFN, MSU Chemistry Department,

Purnachandra Keshavrao Talwalker

and Hunt-Hoppert inbred strain). 3—methylcholanthrene and
9, 10-dimethyl-l, 2—benzanthracene were more effective in
inducing mammary tumors in Sprague—Dawley strain than, 3,

A-beanyrene.

CENTRAL NERVOUS SYSTEM INHIBITION OF
PROLACTIN SECRETION
AND
EFFECT OF ANTERIOR PITUITARY HORMONES
ON INDUCTION OF NORMAL AND NEOPLASTIC

MAMMARY GROWTH IN THE RAT

By

Purnachandra Keshavrao Talwalker

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Physiology and Pharmacology

1964

Dedicated
to my parents
Shri Keshavrao
and

Sou. Kamalabai

ACKNOWLEDGMENT

The author feels highly grateful to Dr. Joseph Meites
for his kind help and guidance throughout the whole course
of his study. His inSpiration, guidance, and encouragement
in finishing this project are greatly appreciated. The
writer also wishes to express his sincere appreciation to
Drs. B. V. Alfredson, E. P. Reineke, J. E. Nellor, W. D.
Collings, and L. F. Wolterink, whose counseling and guidance
were of real value to the author.

Many thanks are due to Dr. C. S. Nicoll, Mr. Albert
Rather, Mr. Roger Deuben, Mr. T. F. HOpkins, Dr. Hideo
Mizuno, and Dr. R. F. Langham (Department of Animal Patho-
logy) for their collaboration in some of these and other
studies. The author also wishes to thank Mrs. Carol Nicoll
and Mr. T. E. Staley for their technical assistance; Dr.

H. R. Hunt (Department of Zoology) for the supply of inbred
rats of Hunt—HOppert strain; and Drs. J. Furth and U. Kim
of Department of Pathology, Francis Delafield HOSpital,
Columbia University, New York, for the supply of pituitary
"mammotropic" tumors.

This work was supported by grants from the National
Institutes of Health, Michigan Cancer Foundation, an insti-
tutional Research Grant from American Cancer Society, and

the Michigan Agricultural Experiment Station.

TABLE OF CONTENTS

INTRODUCTION

EXPERIMENTAL

Section One

 

Central Nervous System Inhibition of
Prolactin Secretion

I. Effects of Chlorpromazine on Mammary Glands of
Rats (Am. J. Physiol. 199: 1073, 1960)

II. In Vitro Inhibition of Pituitary Prolactin
Synthesis and Release by Hypothalamic Extract
(Am. J. Physiol. 205: 213, 1963)

Addendum:

A. Effect of Bradykinin on Pituitary
Prolactin Release In Vitro

B. Effect of Dialyzed_Rat Hypothalamic
Extract on Pituitary Prolactin Release
In Vitro

C. Effects of Hypothalamic Acid Extracts
from Different Species on Pituitary
Prolactin Release In Vitro

III. Ingivo and In Vitro Prolactin Secretion by
TranSplanted Rat ”MammotrOpic” Pituitary Tumors

Addendum:

A. Prolactin Assays of Different Pituitary
”Mammotropic” Tumor Strains

B. TranSplantation of Pituitary "MammotrOpic”
Tumor, Strain F4, into Sprague—Dawley Rat

II.

III.

IV.

APPENDIX

Section Two

 

Effect of Anterior Pituitary Hormones on
Induction of Normal and NeOplastic Mammary
Growth in the Rat

Mammary Lobulo-Aveblar Growth in Adreno-
Ovariectomized Rats Following TranSplantation
of a "Mammotropic" Pituitary Tumor

Induction of Mammary Lobulo-Alveolar Growth by
Anterior Pituitary Hormones in Adreno—Ovaricetomized
Rats (Proc. Soc. Exp. Biol. and Med. 107: 880, 1961)

Mammary Tumor Induction by Estrogen or Anterior
Pituitary Hormones in Ovariectomized Rats Given
7, l2-Dimethyl-l, 2-Benzathracene

Addendum: Induction of Tumors in Different Strains
of Rats by Intramammary Administrations of Chemical
Carcinogens

Curriculum Vitae and List of Published Papers

INTRODUCTION

One of the characteristics of humans is the desire
to simplify the complexities of nature by generalized ex-
planations. Primitive man invoked Spirits and demons to
explain natural phenomena. With the advent of science,
such explanations have become unsatisfactory and we now
insist on more mechanistic hypotheses that can be experi-
mentally verified. The value of a theory, hypothesis or a
concept must be judged either in terms of variety and type
of observations that can be accounted for, or by its effec-
tiveness in stimulating research and influencing the design
of experiments.

Recently in endocrinology, as a result of studies in-
volving central and peripheral nerve lesions, pituitary stalk
section, hypOphyseal tranSplants, electrical stimulation,
neurohumoral stimulants, pharmacological agents and in vitrg_
cultures of pituitary tissue, the concept has developed that
the central nervous system (CNS), and particularly hypothalamus
is essential for normal secretion of 5 of the anterior pitui-
tary hormones, but it inhibits prolactin secretion by way of
its neurovascular linkage to the pituitary (l).

PsychotrOpic agents such as chlorpromazine, which sup—
presses the CNS, evoke galautorrhea in women and induce pseudo-
pregnancy in the rat, indicating release of pituitary prolactin

(2, 3). This suggests that chlorpromozine overcomes CNS

inhibition to pituitary prolactin secretion, and thereby
fosters its release. The effect of chlorpromozine on mammary
growth and secretion in the rat was therefore investigated.

Hypothalamic inhibition of pituitary prolactin release
is probably mediated through release of a hypothalamic
factor(s) which is tranSported through the hypothalamo-
hypOphyseal portal system to the anterior-pituitary. Such a
concept requires the demonstration of the existence of a
prolactin inhibiting factor (PIF) in the hypothalamic tissue.
Therefore, it was decided to determine whether homogenates
or acid extracts of rat hypothalamus could inhibit pituitary
prolactin secretion, and whether such inhibition could be
due to one of the known neuropharmacological agents present
in the hypothalamus.

Removal of hypothalamic inhibition to the pituitary,
by transplanting it from its normal cranial site to other
parts of the body, or by culturing it.l2.l£§£2: fosters pro-
lactin release (1). In some strains of rats and mice, the
pituitary tranSplant may grow into a tumor and secrete large
amounts of prolactin as indicated by mammary gland stimulation
and a pseudOpregnancy reSponse in the rat (4-6). The rat
"mammotropic" pituitary tumor develOped by Furth, similarly
induces mammary gland stimulation in the host rat (7). The
functional behaviour of this pituitary tumor appears to be
analogous to a normal pituitary removal from its hypothalamic

connections, insofar as prolactin secretion is concerned.

Therefore, the capacity of this pituitary tumor to elaborate
prolactin ig vivo and in vitro was investigated, and this
was compared with prolactin secretion by normal rat pituitary

in situ.

 

In the second part of this thesis, effect of anterior
pituitary hormones on normal and neOplastic mammary growth
were studied. The importance of estrogen for the deveIOp-
ment of the mammary lobulo-alveolar (L-A) system is well
established. Estrogen stimulate mammary L-A growth in intact
rats and mice, but is ineffective in the absence of the
anterior pituitary. Since estrogen can increase pituitary
prolactin and probably STH secretion, this suggests that
estrogenic stimulation of mammary L-A growth is probably
mediated in part through increasing prolactin and STH secre—
tion (1).

Clifton and Furth (8) observed that in adreno-gonadect-
omized rats, mammary L-A growth was induced by tranSplanting
a pituitary tumor which produces large amounts of prolactin
STH and ACTH. Such a tumor provided a continuous source of
these hormones in increasing concentrations as the tumor grew
in the hosts. These workers did not check for completeness
of adreno-gonadectomy, and histological examination of the
mammary glands was not reported. It was thought of interest
therefore, to re-examine and extend this study in adreno-
ovariectomized rats, and examine the evidence for estrogenic

activity and completeness of adrenalectomy.

The results showed that in the apparent absence of
estrogens, the hormones (prolactin, STH and ACTH) secreted
by the pituitary tumor stimulated mammary L-A growth in
adreno-ovariectomized rats° This emphasized the importance
of prolactin and STH in mammary growth in the rat. It was
decided therefore, to determine whether frequent injections
of large doses of pituitary hormones (thus mimicking the
effects of the pituitary tumor tranSplant) could stimulate
mammary L-A growth in adreno-ovariectomized—hyp0physectomized
rats.

Studies involving mammary carcinogenesis in the rat
suggested that mammary tumorogenesis consists of a two-phase
mechanism: (a) an initiating phase in which a carcinogen
produces an irreversible alteration in the cells of the
mammary gland, and (b) a promoting phase in which hormones
stimulate growth of these altered cells (7). Although pitui—
tary hormones maintain or stimulate growth of already estab-
lished mammary tumors in rats, it is not clear whether hormones
participate in the initiating phase of mammary carcinogenesis
by a chemical carcinogen. Therefore, the role of estrogen
and of prolactin and STH during the initiating phase of mammary
tumorogenesis by a chemical carcinogen was investigated.

Experiments I, II and V in this thesis have already
been published, while all other experiments reported here

are as yet unpublished.

REFERENCES

Meites, J., Nicoll, C. S. and Talwalker, P. K. in

Advances in Neuroendocrinology (A. V. Nalbandov ed.),

 

University of Illinois Press, Urbana, 1963, Chapt. 8,

p. 238.

Barraclough, C. A. and C. H. Sawyer. Endocrinology.

65: 553, 1959.

Sulman, F. G. and H. Z. Winnik. Nature, 178: 365, 1956.

 

Gardner, W. U. in On Cancer and Harmones (C. Huggins ed.),

 

University of Chicago Press, Chicago, 1963, p. 89.
Mfihlbock, 0. and Boot, L. M. Cancer Research, 19: 402,

1959.
Kullander, S. Cancer Research, 20: 1079, 1960.

 

Furth, J. Fed. Proc., 20: 865, 1961.

 

Clifton, K. H. and Furth, J. Endocrinology, 66: 893,

 

1960.

Section One

 

CENTRAL NERVOUS SYSTEM INHIBITION OF

PROLACTIN SECRETION

I. EFFECTS OF CHLORPROMAZINE ON'

MAMMARY GLANDS OF RATS

ABSTRACT

The effects of chlorpromazine were determined on
mammary growth and initiation of milk secretion in virgin
rats, and on maintenance of mammary structure and secretion
in postpartum rats after litter removal. When chlorpromazine
was administered in doses of 5 or 15 mg/kg body weight for 5
days, only the higher dose was effective in inducing lobulo-
alve01ar growth and initiating milk secretion in rats
initially primed with 10 ug estradiol daily for 10 days. The
higher dose of chlorpromazine also maintained mammary lobulo-
alveolar structure and secretion in postpartum rats for 10
days after litter removal. Chlorpromazine produced a sign-
nificant increase in adrenal weight and decrease in thymus
weight, indicating adrenal stimulation. In estrogen-primed
rats neither prolactin nor ACTH alone could initiate mammary
secretion; however they were effective when given in combin-
ation. The present data indicate that chlorpromazine promotes
the secretion of both prolactin and ACTH. HypOphysectomy
following estradiol treatment prevented chlorpromazine from
initiating mammary secretion, showing that its effects are
mediated through the anterior pituitary and not directly on

the mammary gland.

Recently attention has been focused on the influence
of several tranquilizing drugs on anterior pituitary function.
Chlorpromazine may inhibit TSH (1) and FSH-LH (2,4) secretion,
and stimulate secretion of ACTH in several Species (3).
Chlorpromazine apparently promotes prolactin secretion as
indicated by induction of pseudOpregnancy, maintenance of
corpora lutea beyond the normal period of pseudopregnancy in
the rat (4) and occasional galactorrhea in women (5,6). It
became of interest to determine whether this drug could induce
mammary growth and secretion in the rat, since these processes
are dependent upon anterior pituitary secretion of prolactin

and ACTH.

METHODS

Virgin femakerats of the Carworth strain, weighing
200-250 gm each, were maintained in a temperature controlled
(74i2°F) and artificially illumined (14 hours per day) room.
They were fed a normal laboratory diet, and food and water
were available ad libitum. Three different experiments were
performed as follows:

Experiment 1: The effects of chlorpromazine on mammary
growth were investigated in virgin rats. Thirty-two rats
were divided into three groups of 12, 10 and 10 each, and
were injected subcutaneously with saline or chlorpromazine
in doses of 5 or 15 mg/kg body weight for 15 days.

Experiment 2: The ability of chlorpromazine to induce

mammary secretion was determined in estrogen-primed virgin

rats. Fifty-five rats were divided into 9 groups and
injected subcutaneously with 10 pg estradiol daily in 0.1 m1
corn oil for 10 days. This treatment induces lobulo-alveolar
(L-A) development in the rat and is necessary to render the
mammary tissues reSponsive to hormones which stimulate milk
secretion (7). For the subsequent 5 days the groups

received subcutaneous injections as follows: (1) controls,
saline 0.1 m1 once daily; (2) and (3), chlorpromazine, 5 and
15 mg/kg body weight, reSpectively, once daily; (4) and (5),
prolactin (Ovine, 20 I.U./mg), 1.0 and 2.0 mg, reSpectively,
twice daily; (6) and (7) ACTH, 1.0 and 2.0 I.U., reSpectively,
twice daily; (8) prolactin, 1.0 mg and ACTH 1.0 I.U., each
twice daily. In the last group (9) rats were hypOphysecto-
mized by the parapharyageal approach after 10 days treatment
with estradiol and then injected with chlorpromazine, 15 mg/kg
body weight, for 5 days once daily. Chlorpromazine and pro-
lactin were dissolved in physiological saline, and ACTH was
injected in a gel-preparation.

Experiment 3: The effects of chlorpromazine on the
maintenance of L—A structure and secretion in postpartum rats
were studied. Thirty rats were bred and placed in individual
cages. 0n the first day following parturition the litters
were reduced to six young each. During the following three
days the litters were weighed daily to check lactational per-
formance of the mother, and on the 4th day the litters were

withdrawn. The lactating rats were then divided into three

groups of 10 each and injected subcutaneously with saline or
chlorpromazine in doses of 5 or 15 mg/kg body weight for the
following 10 days.

The day after the last day of treatment the rats were
sacrificed and the right inguinal mammary glands were removed
and prepared for histological examination by standard tech-
niques (7). ,The ovaries, uterus, adrenals and thymus were
excised from the animals of Experiment 1, and weighed on a

Roller-Smith balance.

RESULTS

Experiment 1: (Table I). The mammary glands of the
saline-treated controls (gr. 1) consisted essentially of a
few ducts and end buds (Fig. 1. For figures see appendix
for published article). The mammary glands from the rats
treated with the lower dose of chlorpromazine (gr. 2) did
not differ from the controls (gr. 1). At the higher dose
level (gr. 3), chlorpromazine induced considerable L-A devel-
Opment (Fig. 2). The weights of the ovaries and uterus from
gr. 2 and 3 were not significantly different from the controls
(gr. 1). However, the average adrenal weights were signifi—
cantly higher (P.< .05) and average thymus weights were sig-
nificantly lower (P<< .01) in gr. 3 than in the controls
(gr. 1).

Experiment 2: (Table II). Estradiol treatment for 10
days induced L—A development but no secretion in virgin

female rats (7). Subsequent treatment with saline for 5 days

(gr..1) resulted in mammary regression to almost a bare duct
system in all rats and no secretion was observed (Fig. 3).
Treatment with the lower dose of chlorpromazine (gr. 2) had
no effect, since the mammary glands regressed to the same
extent as in the controls (gr. 1). However, treatment with
the higher dose of chlorpromazine (gr. 3) induced maintenance
of L-A structure and induction of secretion in all rats of
this group (Fig. 4).

Experiment 3: (Table III). In postpartum rats after
removal of litters and treatment with saline for 10 days
(gr. 1), the mammary glands regressed from a secretory L-A
structure to ducts with a few small closed alveoli and no
secretion (Fig. 8). Mammary glands from rats treated with
the lower dose of chlorpromazine (gr. 2) did not differ
histologically from the controls (gr. 1). Mammary involution
was markedly inhibited in the rats treated with the higher
dose of chlorpromazine (gr. 3). L—A structure and some
secretion were maintained in 9 out of 10 rats (Fig. 9).

In order to determine whether the effects of chlorpro-
mazine were mediated through prolactin alone or also required
ACTH, the two hormones were administered individually and in
combination to estradiol-primed rats. When prolactin was
administered at dose levels of 1 or 2 mg twice daily it pro-
duced maintenance of L-A structure but no secretion (Fig. 5).
When ACTH was injected at dose levels of 1 or 2 I.U. twice

daily, there was no secretion and the mammary glands regressed

Ill {1.}.

III) 4I|IIIIII|E[ Ail! (ill-[I l‘rlllllll.[l|‘ z'llr‘llllulll'll‘ fli‘l’ll

(Fig. 6) as in the controls. However, when both prolactin
and ACTH were administered, 4 out of 5 rats showed mammary
secretion and maintenance of L-A structure (Fig. 7). Hypo-
physectomy following estradiol treatment, and subsequent
treatment with chlorpromazine at the higher dose level,
resulted in complete regression of L—A structure. This
indicates that the effects of chlorpromazine are mediated

through the pituitary.

DISCUSSION

The results of the present study show that an appro-
priate dose of chlorpromazine in the rat can (a) induce L-A
growth (b) initiate mammary secretion after estrogen-priming
and (c) retard mammary involution following litter removal
in postpartum rats. L-A develOpment is believed to be
induced in virgin rats by increased secretion of prolactin
and stimulation of ovarian luteal function. HypOphysectomy
following ostradiol treatment prevented chlorpromazine from
initiating mammary secretion, showing that its action is
mediated through the anterior pituitary and not directly on
the mammary gland. Both prolactin and ACTH were necessary
to initiate mammary secretion in catrogen-primed rats.
Neither hormone was effective alone. The rat is apparently
exceptional in this reSpect, since prolactin alone can initi-
ate mammary secretion in intact animals of other Species with

develOped mammary glands (8).

In postpartum rats, maintenance of mammary structure
and secretion is partially dependent upon the suckling
stimulus which has been shown to induce release of both pro-
lactin andiKflEIfrom the anterior pituitary (8). Mammary
involution which normally follows litter removal or weaning
can be partially inhibited by administration of prolactin
or cortisone (9). In hypOphysectomized rats, milk secretion
can also be partially maintained by injecting prolactin and
ACTH (10). Maintenance of L-A structure and secretion in
postpartum rats with chlorpromazine in the present experi-
ment is therefore believed to be due to increased secretion
of both prolactin and ACTH.

Chlorpromazine has been reported to induce ACTH release
in several Species (3). In the virgin female rats treated
for 15 days with chlorpromazine, there was a significant
increase in adrenal weight and decrease in thymus weight.
Multiple injections of chlorpromazine may induce maximum
secretion of ACTH in rats, as has been reported for reserpine
(11). Stimulation of prolactin secretion by chlorpromazine
is indicated by its ability to induce pseudOpregnancy, main—
tain corpora lutea beyond the normal period of pscudOpreg-
nancy in rats (4), evoke galactorrhea in women (5,6) and in
the present study, initiate mammary secretion in estrogen-
primed rats. Preliminary assays in our laboratory of pitui-
taries from postpartum rats following chlorpromazine adminin-

istration suggests that prolactin is released.

Recently, it has been proposed that factors which sup-
press FSH-LH secretion promote the secretion of prolactin
(12). Chlorpromazine has been reported to inhibit ovulation
in the rat in reSponse to mechanical stimulation of the
uterine cervix or injection of estradiol when administered
during estrus (2). There is also evidence that factors which
promote the secretion of ACTH suppress LH secretion (13).

It has been observed that non-Specific stresses, drugs and
emotional disturbances inhibit ovulation and at the same
time promote ACTH secretion (14, 15). Harris (16) concluded
that most conditions which promote ACTH secretion also
inhibit TSH secretion. Chlorpromazine and reserpine have
been reported to inhibit TSH secretion (1,17). It is possi-
ble therefore, that some of the factors which promote the
secretion of ACTH and prolactin also suppress FSH, LH and
TSH secretion by the anterior pituitary.

Recent studies have shown that tranSplantation of the
anterior pituitary from its normal cranial site to other
parts of the body favors secretion of prolactin (18). In
our laboratory we have observed that pituitary implants
underneath the kidney capsule secrete sufficient prolactin
to initiate mammary secretion in estrogen-primed rats (19).
It has been suggested that the hypothalamus may normally
inhibit prolactin secretion by way of its neurovascular
linkage to the anterior pituitary (l8). Barraclough and

Sawyer (4) suggested that reserpine and chlorpromazine

depress the hypothalamus and thus remove inhibition to pro-
lactin secretion. Studies of the effects of chlorpromazine
on ACTH secretion indicate that its action is at the pre-
pituitary and probably at the hypothalamic level (3,20).
The ability of chlorpromazine and various other factors
such as the suckling stimulus (8), adrenalectomy (21),
numerous drugs, electrical stimulation, and nonSpecific
stresses (22-25) to induce secretion of prolactin and ACTH
suggests the existence of a common mechanism(s) regulating

the secretion of these two hormones.

10.

ll.

l2.

l3.
l4.

15.

16.

REFERENCES

Mayer, S. W., F. H. Kelly, and M. F. Morton. J. Pharm-
acol. and Exper. Therap. 117: 197, 1956.

Alloiteau, J. J. C. R. Soc. de Biol. 151: 207, 1957.
Woodbury, D. M. Pharm. Rev. 10: 275, 1958.

Barraclough, C. A. and C. H. Sawyer. Endrocinology 65:
563, 1959.

Polishuk, W. Z. and S. Kalcsar. J. Clin. Endoc. and
Metab. 16: 292, 1956.

Sulman, F. G. and H. Z. Winnik. Nature 178: 365, 1956.
Meites, J. Proc. Soc. Exp. Biol. and Med. 100: 750, 1959.
Meites, J. In: Reproduction in Domestic Animals, edited
by H. H. Cole and P. T. Cupps. New York: Acad. Press,
1959, p. 539.

Meites, J. and C. S. Nicoll. Endrocinology 65: 572, 1959.
Lyons, W. R., C. H. Li, and R. E. Johnson. Recent Prog.
in Hormone Research 14: 219, 1958.

Kitay, J. 1., D. A. Holub, and J. W. Jailer. Endrocrin-
ology 65: 548, 1959.

Rothchild, I. Endrocinology In press.

Selye, H. Endrocrinology 25: 615, 1939.

Seyle, H. Annual Report on Stress. Montreal: Acta,
Inc. 1951, p. 120.

Harris, G. W. Neural Control of the Pituitary Gland.
London: Edward Arnold, Ltd. 1955, p. 70 and 169.

ibid. p. 145.

IO

17. Moon, R. C. and C. W. Turner. Proc. Soc. Exp. Biol.
and Med. 102: 134, 1959.
18. Everett, J. Endrocinology 54: 685, 1954.
19. Meites, J. and T. F. HOpkins. Proc. Soc. Exp. Biol.
and Med., 104: 263, 1960.
20. Mahfouz, M. and E. H. Ezz. J. Pharmacol. and Exper.
Therap. 123: 39, 1958.
21. Swingle, W. W., E. J. Fedor, G. Barlow, E. J. Collins,
and J. Perlmutt. Am. J. Physiol. 167: 593, 1951.
22. Meties, J., C. S. Nicoll, and P. K. Talwalker. Proc.
Soc. Exp. Biol. and Med. 101: 563, 1959.
23. Meites, J., C. S. Nicoll, and P. K. Talwalker. Proc.
Soc. Exp. Biol. and Med. 102: 127, 1959.
24. Nicoll, C. S., P. K. Talwalker, and J. Meites. Am. J.
Physiol. 198: 1103, 1960.
25. Swingle, W. W., P. Seay, J. Perlmutt, E. J. Collins,
G. Barlow, E. J. Fedor. Am. J. Physiol. 167: 586,
1951.
FOOTNOTES
1. Published with the approval of the Michigan Agricultural
Experiment Station as Journal Article No. 2657.
2. This investigation was supported in part by a grant from
the National Institutes of Health (C-3448) to J. Meites.
3. We wish to express our appreciation to Dr. S. Scheidy,

Smith Kline, and French Laboratories, Philadelphia, for

ll

chlorpromazine; Endrocrinology Study Section, NIH, for

prolactin; Dr. R. C. Stafford, The Upjohn Co., Kalamazoo,

Mich., for ACTH; and Dr. J. 0. Reed, Foundation Labora-

tories, New York, for estradiol.

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II. IN_VITRO INHIBITION OF
PITUITARY PROLACTIN SYNTHESIS AND RELEASE

BY HYPOTHALAMIC EXTRACT

ABSTRACT

Anterior pituitaries from rats were incubated for 2 hours
at 37.5°C in a Dubnoff metabolic shaker in an athSphere of
95% 02 - 5% C02. At the end of the indubation period, 169%
more prolactin was found in the medium and anterior pituitar-
ies than in fresh non—incubated anterior pituitary, demon-
strating that net synthesis and release of prolactin had
occurred. When anterior pituitaries were incubated together
with homogenate or acid extract of rat hypothalamus, prolactin
levels in the medium andanterior pituitaries were markedly
decreased, indicating inhibition of synthesis and release.
Acid extract of rat cerebral cortex had no effect on pituitary
prolactin synthesis or release. Incubation of purified ovine
prolactin or rat prolactin, with or without homogenate or
acid extract of rat hypothalamus, did not alter prolactin
activity, demonstrating that the inhibitory action of homo-
genate or acid extract of the hypothalamus was not due to
inactivation of prolactin. Acetylcholine iodide, epinephrine,
l-norepinephrine bitartrate, serotonin creatinine sulfate,
histamine acid phOSphate, oxytocin (Pitocin), synthetic argin-
ine and lysine vasopressin, and substance P had no effect on
pituitary prolactin release in 31339, The results indicate
that the hypothalamus contains a factor(s) which inhibits

synthesis and release of prolactin by the anterior pituitary

lg vitro, and this factor(s) does not appear to be any of the

known neurohumoral agents in the hypothalamus.

Recent studies have emphasized that the CNS and particu-
larly the hypothalamus participate in the control of anterior
pituitary function. It has been observed that placement of
hypothalamic leisons, transection of the pituitary stalk,
tranSplantation of the anterior pituitary to non-cranial
sites, use of certain CNS depressant drugs, and organ cultures
of the anterior pituitary are accompanied by a marked decrease
in secretion of all anterior pituitary hormones except pro-
lactin. Under these conditions, prolactin secretion is
actually increased, as indicated by prolonged maintenance of
the corpus luteum in the rat, induction of mammary growth and
lactation, and increased secretion of prolactin by the pitui-
tary in 31339 (1,2). These observations suggested that the
hypothalamus may contain a factor(s) which normally inhibits
prolactin secretion. Presumably this factor is transported
through the hypothalamico-hypophyseal portal system to the
anterior pituitary, where it acts to depress prolactin synthe-
sis and/or release.

This investigation was undertaken to determine whether
homogenates or acid extracts of rats hypothalamus could
inhibit prolactin secretion, and whether such inhibition could
be due to one of the neurOpharmacological agents known to be
present in the hypothalamus. Short term incubation of the
anterior pituitary was adOpted as a test method (3), since
under these conditions the anterior pituitary is isolated from

its normal neurovascular linkage and is removed from all body

influences. A preliminary report of the present work has

previously been published in abstract form (4).
METHODS AND RESULTS

Animals. Mature virgin female Carworth rats of the
CFN strain, 3-5 months old, were used as pituitary donors
in all experiments. Rats were maintained in a temperature
controlled (75 :_1°F) and artificially illuminated (14 hrs/
day) room. They were fed ad_libitum on Wayne Lab Blox
pellets. White King squabs, 5-8 weeks old, were used for
prolactin assays. A total of 820 rats and 805 pigeons were

used in this study.

Incubations. The rats were first stunned and then de-

 

capitated. The anterior pituitary was separated from the
posterior lobe and placed in a Petri dish over filter paper
moistened with synthetic protein-free medium ”199"3 (5).

Each anterior pituitary was cut into half, weighed, and trans-
ferred to a 25 ml pyrex flask containing medium ”199” (pH 7.4).
A total of 6 or 12 anterior pituitary halves were placed in
each flask. All incubations were carried out in a Dubnoff
metabolic shaker (60 cycles per minute) under constant gassing

with humidified 95% 02 - 5% cog, at 37.5 : o.5°c.

Prolactin Assays. Prolactin activity of the samples

 

was determined by the intradermal pigeon crop assay method

using a paired assay procedure. The control sample was

injected over one side of the crop sac and the experimental
sample over the other side in the same pigeons. This provided
a direct comparison between the two samples. Anterior pitui-
taries released considerable quantities of prolactin into

the medium during incubations. Therefore, the medium was
injected without any further treatment, while the anterior
pituitaries were homogenized with medium "199” and injected
as such. Prolactin activity was expressed as IU per 100 mg
of anterior pituitary incubated. The details of the assay
procedure and statistical treatment have been described
elsewhere (6).

Experiment 1. Synthesis and Release of Prolactin
In Vitro

 

Anterior pituitaries from groups of 3 rats each were
divided into halves, weighed, transferred to a flask contain-
ing 2 ml of medium "199", and incubated for 2 hours. At the
end of this period, the medium and the incubated anterior
pituitaries were separated and assayed independently for
prolactin activity. In order to determine initial prolactin
content, anterior pituitaries from either 5 or 6 rats of the
same age group were removed at the same period, pooled,
weighed and assayed for prolactin activity.

The results are summarized in Table 1. An average of
0.8 IU of prolactin was released into the medium per 100 mg
of anterior pituitary (AP) incubated. Anterior pituitaries

at the end of indubation contained an average of 2.7 IU

prolactin per 100 mg AP. The prolactin content of the medium
and the incubated AP together was 3.5 IU per 100 mg AP as
compared with 1.3 IU per 100 mg AP present at the beginning
of the experiment. This represents a 169% increase in pro-
lactin over that contained in freshly excised AP. The
results of this experiment show that net synthesis and
release of prolactin can occur during 2 hours of incubation.
We have also observed a gradual increase in the total amount
of prolactin released into the medium during a 4 hour period
of incubation (unpublished).

Experiment 2. Effect of Homogenate of Hypothalamus
on Prolactin Secretion In Vitro

 

 

Anterior pituitaries from 6 rats were cut into halves,
weighed and placed into 2 flasks, each containing 2 ml of
medium "199". Each flask contained 6 halves from the same 6
pituitaries. Preliminary studies showed that under these
conditions, the incubated anterior pituitaries released the
same amounts of prolactin in both flasks. The hypothalamus
(including the median eminence) from the same 6 rats was
homogenized and added to the experimental flask, and an
equivalent volume of medium "199" was added to the control
flask. At the end of 2 hours incubation, the medium and the
incubated pituitary halves were removed from the control and
experimental flasks, and assayed against each other in the
same pigeons for prolactin activity.

The results are summarized in Table 2. When anterior

pituitaries were incubated with rat hypothalamic homogenate,

significantly less prolactin was released into the medium in
all 14 flasks than in the correSponding control flasks not
containing hypothalamic homogenate. An average inhibition
of prolactin release of 75.4% was observed. Similarly, the
prolactin content of the anterior pituitary halves incubated
together with hypothalamic homogenate was significantly less
(51.8%) than in the correSponding control anterior pituitary
halves. These results show that hypothalamic homogenate can
inhibit prolactin synthesis and release in_yit§3,

Experiment 3. Effect of Acid Extracts of Hypothalamus

and Cerebral Cortex on Prolactin
Secretion In Vitro

 

 

 

 

The hypothalami from 36 rats were removed, pooled,
weighed, homogenized with 2.4 m1 0.1N H01, and centrifuged
(12000 x G) for 30 minutes at 4°C. The supernatant was
added to medium ”199" and the pH was adjusted to 7.4 by
adding NaHCO3 solution. The final volume was made up so
that each m1 of medium contained the acid extract from 3 rat
hypothalami. Equivalent amounts of acid extract of cerebral
cortex were similarly prepared. To each of the experimental
flasks, 2 ml of the medium containing acid extract of hypo-
thalamus or cerebral cortex was added, and to each of the
correSponding control flasks, 2 m1 of medium ”199" was
similarly prepared but without any tissue extract. At the
end of the incubation period, the medium and the incubated
anterior pituitary halves were removed and assayed separately

for prolactin activity.

The results are summarized in Table 3. When 3 rat
anterior pituitaries were incubated with acid extract from
4 rat hypothalami, an average of 54% less prolactin was
found in the medium and 36% less in the incubated anterior
pituitaries reSpectively than in the correSponding controls.
Greater inhibition (72% and 61%) of prolactin release into
the medium and of anterior pituitary prolactin content (68%
and 56%) was observed when anterior pituitaries were incu-
bated with acid extract from 6 rat hypothalami. Acid
extract of cerebral cortex had no effect on either prolactin
release into the medium or anterior pituitary prolactin con-
tent. These results demonstrate that hypothalamic acid
extract can inhibit synthesis and release of prolactin by
the anterior pituitary EB;XEE£2-

Experiment 4. Effect of Incubation per se on
Prolactin Activigy

 

 

 

‘A. A standard NIH ovine prolactin (15 IU/mg) prepara-
tion was added to 2 m1 of medium ”199” in each of 6 flasks.
Three flasks were incubated for 2 hours, and 3 other flasks
served as controls and were not incubated. At the end of 2
hours, pooled medium from the control flasks was assayed
against pooled medium from the experimental flasks.

B, Anterior pituitaries from 6 rats were divided into
halves, transferred to a flask containing 4 ml of medium
"199", and incubated for 2 hours. At the end of incubation,

the anterior pituitary halves were removed and the medium

was equally divided and placed into 2 flasks. The medium
in each control flask was not incubated, while the medium
in each experimental flask was incubated again for 2 hours.
Media from 3 control and 3 experimental flasks were each
pooled and assayed for prolactin activity.

The results are summarized in Table 4. No significant
difference was observed in prolactin content between incubated
and non-incubated medium containing NIH ovine prolactin.
Similarly, incubation had no effect on rat pituitary prolactin.
This shows that under the above experimental conditions,
incubation.£§r_§§_neither activates nor inactivates NIH ovine
or rat prolactin.

Experiment 5. Effect of Homogenate or Acid Extract

of Hypothalamus on Activity of
Prolactin Preparations

 

 

 

 

A, Standard NIH ovine prolactin preparation was added
to flasks containing 2 ml of medium ”199" containing acid
extract from 6 rat hypothalami. At the end of 2 hours incu-
bation, the medium from the 3 control flasks and the 3
experimental flasks were pooled and assayed for prolactin
activity.

B, Anterior pituitaries from 6 rats were divided into
halves, transferred to a flask containing 4 m1 of medium
"199", and incubated for 2 hours. At the end of incubation,
the medium was separated from the pituitary halves, and was

divided equally and placed into 2 flasks. Homogenate or acid

extract from 6 rat hypothalami was added to an experimental

flask, and equivalent amounts of medium "199” was added to
the control flask. Each pair of flasks was further incubated
for 2 hours, and the medium was assayed separately, or by
combining media from either 3 control or the correSponding
3 experimental flasks.

The results in Table 5 indicate that (a) incubation of
NIH ovine prolactin with rat hypothalamic acid extract does
not activate or decrease prolactin activity (b) the activity
of rat pituitary prolactin released into the medium is not
increased or diminished when incubated with hypothalamic
homogenate or acid extract of hypothalamus and (c) homogenate
or acid extract of hypothalamus does not modify the pigeon
crop reSponse to prolactin.

Experiment 6. Effects of Neuropharmacological Agents
on Prolactin Release In Vitro

 

 

 

Anterior pituitaries from 6 rats were cut into halves,
weighed and placed into 2 flasks, each containing 2 ml of
medium "199”. Each of the two flasks contained 6 halves from

the same 6 pituitaries. Acetylcholine iodide,br epinephrine,“

l-norepinephrine-bitartrate,5 serotonin creatinine sulfate,6
histamine acid phOSphate,7 substance P (9U/mg), oxytocin
(Pitocin8), synthetic lysine vasopressin,9 and synthetic
arginine vasopressin9 were added to experimental flasks,
while the correSponding control flasks contained an equivalent

volume of medium "199”. Solutions of these agents were

brought to pH 7.4 before placing them into the flasks. The

dosages of these agents are given in Table 6. Each agent
was added once at the beginning and again one hour after
incubation was begun.

At the end of 2 hours incubation, the media from the
control and the experimental flasks were removed and assayed
against each other for prolactin activity. In most cases,
media from 3 control flasks and the correSponding 3 experi-
mental flasks were pooled separately and assayed, with the
exception of the incubations with acetylcholine iodide,
epinephrine and serotonin creatinine sulfate. In these, the
medium from each of the flasks was assayed separately. The
commercial preparation of oxytocin (Pitocin) solution con-
tained 0.5% chlorobutanol as a preservative. Therefore, an
equivalent amount of chlorobutanol was added to the control
flask.

The results (Table 6) Show that incubation of anterior
pituitary with all the neurOpharmacological agents employed,
at the dose levels used, had no significant effect on pro-
lactin release into the medium. This indicates that the
hypothalamic inhibition of anterior pituitary prolactin
release in vitro is not due to any of the above neurOpharma-
cological agents.

Experiment 7. Effects of Neuropharmacological Agents

on Pigeon CrOp Response to NIH OVine
Prolactin

 

 

 

 

This experiment was carried out to determine whether

the neurOpharmacological agents employed above could modify

10

the pigeon crOp reSponse to NIH ovine prolactin by acting
at the crOp level. Standard NIH ovine prolactin was added
to medium ”199". The medium was divided into 2 equal halves,
and acetylcholine iodide, epinephrine, serotonin creatinine
sulfate, histamine acid phOSphate, oxytocin (Pitocin) or
Pitressin8 was added to each half in the amounts shown in
Table 7. The correSponding control halves were mixed with
an equivalent volume of medium "199". Both the control and
the experimental media were assayed in the same pigeons for
prolactin activity. The results (Table 7) show that these
neurOpharmacological agents, at the dose levels used, did
not modify the pigeon crop reSponse to NIH ovine prolactin.
Experiment 8. Tests for Prolactin Activity in

Homogenates and Acid Extracts of
Hypothalamus and Cerebral Cbrtex

 

 

 

 

The hypothalamus from 6 rats were homogenized with 2

ml of medium ”199”. The homogenate was injected into 5
pigeons (dose 0.1 ml of homogenate/pigeon/day for 4 days).
Media "199" containing acid extracts of hypothalamus or
cerebral cortex were prepared as described in Experiment 3
and assayed separately for prolactin activity in 10 pigeons
in doses of 0.1 ml of medium/pigeon/day for 4 days. At the
dose levels used, no prolactin activity was detected in
homogenate or acid extracts of hypothalamus or cerebral

cortex.

11
DISCUSSION

The present study shows that synthesis and release of
prolactin by the isolated anterior pituitary can occur
during a 2 hour period of incubation, and that these processes
are inhibited by an homogenate or acid extract of rat hypoth-
alamus. An acid extract of cerebral cortex had no effect on
prolactin synthesis or release, suggesting that this portion
of the brain does not contain the active factor. Inhibition
of prolactin secretion cannot be attributed to proteolytic
descruction of prolactin, since incubation of ovine or rat
hypothalamus did not decrease the biological activity of
these preparations. Also, homogenate or acid extract of rat
hypothalamus did not influence the pigeon crOp reSponse to
prolactin.

The prolactin inhibiting factor(s) does not appear to
be any of the known neuropharmacological substances present
in the hypothalamus (7, 8), since incubation of the anterior
pituitary with acetylcholine iodide, epinephrine, 1-norepine-
phrine bitartrate, serotonin creatinine sulfate, substance P,
oxytocin (Pitocin) or synthetic lysine and arginine vaso-
pressin at the dose levels used, had no effect on prolactin
release. Previously, we reported that injections of acetyl-
choline, epinephrine, norepinephrine or serotonin into
estrogen-primed rats or rabbits initiated mammary secretion
(9—11). The present study shows that the action of these

drugs in stimulating prolactin secretaion is not by way of a

12

direct action on the anterior pituitary but through other
mechanisms. It is possible that they act by inhibiting

synthesis or release of the prolactin inhibiting factor(s)
of the hypothalamus. It has been suggested that oxytocin
may be reSponsible for inducing prolactin release from the

anterior pituitary (12), but most evidence from in vivo

 

and in 11339 studies (13, 14) does not support this view.
Acid extracts of the hypothalamus apparently contain
the FSH, LH, TSH, and ACTH releasing factors, as well as
prolactin inhibiting factor(s). Intrapituitary infusions
of acid extracts of median eminence tissue have been reported
to induce release of gonadotropin as determined by ovulation
in the rat or the rabbit (15, 16). GonadotrOpic activity of
the medium from anterior pituitary cultures was markedly
increased following addition of acid extract of the hypoth-
alamus to a culture medium (17). An acid extract of rat
stalk median eminence tissue elicited LH release in rats as
measured by ovarian ascorbic acid depletion (18). An acid
extract of bovine median eminence tissue and a polypeptide
extracted from dog hypothalamus stimulated TSH release (19,
20). Similarly, injections of acid extracts of hypothalamus
stimulated ACTH release from the pituitary in the rat (21).
In all these studies, acid extracts of cerebral cortical
tissue were ineffective. The chemical nature of these hypo-
thalmaic factors is not presently well defined, although
the corticotropin and TSH releasing factors apparently are

polypeptides (22-24).

13

Short term incubation of the anterior pituitary, as
originally described by Saffran and Schally (3), has been
used by many workers for studying release or synthesis of
anterior pituitary hormones such as ACTH (25, 26), TSH (23,
24) and FSH (27). Thus incubation of beef anterior pitui-
tary slices with S35 labelled methionine resulted in incor-
poration of the labelled amino acid into prolactin (28).
When rat pituitaries were incubated with Ola-phenylalanine,
the rate of incorporation into ACTH was found to be linear
for 4 hours (29). After incubation of female rat anterior
pituitaries for 2 hours, the total quantity of FSH present
in medium and incubated pituitary together was found to be
significantly more than in the control anterior pituitaries
which were not incubated (27). It is possible that under
our experimental conditions, incubation of anterior pitui-
tary might have resulted in activation of prolactin in the
pituitary from an inactive form, or that activation and
synthesis might have occurred together. Arguments in favor
of such activation have been presented for prolactin (30)
and for ACTH (31), but no direct proof is presently available
to support this View.

Certain bovine STH and ovine prolactin preparations
have been reported to undergo degradation upon standing in
alkaline buffers (32). However, incubation per_§g_of NIH
ovine prolactin or of rat pituitary prolactin in the present

study did not result in any detectable loss of activity.

14

Similarly, no evidence for destruction of rat ACTH was
observed during a one hour period of incubation nor when
ACTH was incubated together with anterior pituitary tissue
(25). Neither activation nor inactivation of rat pituitary
FSH released into the medium was observed during 2 hours of
incubation (27). The presence of proteinase in a purified
preparation of sheep ICSH (LH) was demonstrated only after
prolonged incubation for 6 to 24 hours (33). In our studies
homogenate or acid extract of hypothalamus did not activate
or inactivate NIH ovine prolactin or rat prolactin. It has
previously been reported that incubation of bovine prolactin
with a variety of rat tissues reduced the biological activity
of the prolactin; however, brain tissue had no effect (34).
It has been reported that proteolytic digestion of bovine
prolactin (35) and STH (36, 37) can be carried out to the
extent of 15 - 29% and 11 - 25%, respectively, without loss
of detectable biological activity. It is possible that under
our experimental conditions limited degradation of prolactin
may have occurred without appreciable loss of biological
activity.

Earlier we reported that in estrogen—primed rats, in-
jections of hypothalamic homogenate from rats, which presum-
ably contains all the factors which stimulate secretion of
the anterior pituitary hormones as well as the prolactin
inhibiting factor(s), initiated mammary secretion (38). It

Should be recognized that initiation of lactation is not a

15

specific reaction to prolactin alone, and is influenced by
other hormones, particularly ACTH and adrenal cortical
steroids. ACTH, cortisone or cortisol have been reported to
initiate mammary secretion in virgin rats, estrogen-primed

or pregnant rats, and rabbits (See 39). The lactation-
inducing effect of hypothalamic homogenate in_yiyg may there-
fore be due to the presence of corticotropin—releasing factor
(CRF), since CRF has been shown to be a potent stimulator

of mammary secretion in estrogen-primed rats (2). In addi-
tion the hypothalamus contains factors which stimulate TSH
(19, 20, 23, 24) and STH (40) secretion by the anterior
pituitary, both of which may exert favorable effects on
mammary secretion (2). The possibility should also be con-
sidered that $2.1lX2 induction of mammary secretion by
hypothalamic homogenate may be nonspecific in nature, since
many nonSpecific agents induce pseudogregnancy (2) or initi-
ate mammary secretion (41).

Several procedures have demonstrated that removal of
hypothalamic regulation of the anterior pituitary results in
increased prolactin secretion, including placement of certain
hypothalamic lesions, pituitary tranSplantation, administra-
tion of certain CNS depressant drugs, and in_vit£2_cultures
of anterior pituitaries (2). The concept has develOped that
the hypothalamus chronically inhibits prolactin secretion by
way of its neurovascular linkage to the anterior pituitary

(42). The results of the present in vitro experiments lend

16

support to this concept and indicate that the rat hypotha-
lamus contains a factor(s) which inhibits prolactin synthesis

and release.

\OCD'fim

10.

ll.

12.

13.

17

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Schrieber, V., M. Rybak, and V. Kmentova. Experientia.

 

17:264, 1961.

Schrieber, V., M. Rybak, and V. Kmentova. Experientia.

 

18:338, 1962.

Saffran, M. Can. J. Biochem. and Physiol. 37:319,

1959-

Guillemin, R. and A. V. Schally. Endocrinology. 65:555,

1959.
van Rees, G. P. Acta Endocrinol. 36:485, 1961.

 

 

 

Kraintz, L. and R. V. Talmage. Fed. Proc. 14:87, 1955.

 

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201:547, 1961.

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Dasgupta, P. R. and F. G. Young. Nature. 189:39, 1958.
Lewis, U. J., J. N. Fisher, and W. P. Vanderlaan.

Programme, 44th Annual Meeting of the Endocrine Society,

 

Chicago, Illinois. 1962, p. 1.

Woods, M. C. and M. E. Simpson. Endocrinology. 68:647,

 

1961.
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White, A., R. W. Bonsnes, and C. N. H. Long. J. Biol.

 

Chem. 143:447, 1942.
Li, C. H., H. Papkoff, P. Fonss-Bech, and P. G. Cond-
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Papkoff, H. Endocrinology. 70:754, 1962°

 

Meites, J., P. K. Talwalker, and C. S. Nicoll. Proc.

 

Soc. Exp. Biol. and Med. 103:298, 1960.

 

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FOOTNOTES

Published with the approval of the Director of the
Michigan Agricultural EXperiment Station as journal
article No. 3122.

This investigation was supported in part by NIH
grant AM-04784—03.

Difco Laboratories, Detroit, Michigan.

Eastman Organic Chemicals, Rochester 3, New York.

K and K Laboratories, Inc., Long Island City 1, New York.
Nutritional Biochemicals Corporation, Cleveland, Ohio.
Eli Lilly & Co., Indianapolis, Indiana.

Parke Davis & Co., Detroit, Michigan.

Sandoz Pharmaceuticals, Hanover, New Jersey.

ACKNOWLEDGMENT

We wish to express our appreciation to Dr. A. V.

Schally of the Department of Physiology, Baylor University

College of Medicine, Houston, Texas, for supplying Subatance

P; Dr. Sidney Gimpel of Sandoz Pharmaceuticals, Hanover, New

Jersey, for synthetic arginine and lysine vaSOpression;

Endocrinology Study Section, National Instiatutes of Health,

for prolactin; and Theordore E. Staley and Roger Deuben for

technical assistance.

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TABLE 4. Effect of 2 Hours Incubation on Prolactin

 

 

 

 

 

 

Activity
Prolactin
No. of No. of IU/ml
Flasks Treatment Pigeons Medium P
NIH Ovine Prolactin
3 Not Incubated 8 0.35:0.08*
>0.20
3 Incubated 8 0.33:0.06
Rat Pituitary Prolactin
IReleased into Medium)
3 Not incubated 8 0.09:0.02
:>0.30
3 Incubated 8 0.10:0.02

 

*Mean.: standard error of mean

 

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II. ADDENDUM

The following additional studies were carried out after

the publication of the preceeding paper.

A. Effect of Bradykinin on Pituitary Prolactin
Release In Vitro

 

Bradykinin is present in various tissues of the body,
including the brain. Since it is found in very high concen-
tration in the hypothalamus (1, 2), it was decided to deter-
mine its effect on prolactin release ip_yit£p, The method
used was the same as described under Experiment 6.

The results of the present experiment (Table II—a)
show that bradykinin, at the dose level used, had no signifi-
cant effect on prolactin release. This would incidate that
bradykinin is not the prolactin inhibiting factor in the
hypothalamus.

B. Effect of Dialyzed Rat Hypothalamic Extract

on Pituitarprrolactin Release In Vitro

An acid extract of rat hypothalamus inhibited pituitary
prolactin release ip_yi££g, as shown in the previous publica-
tion. It was decided, therefore, to determine the effect of
dialysis on prolactin inhibiting activity by rat hypothalamic
extract. The method used was the same as described under
Experiment 3. Rat hypothalamic acid extract was neutralized
with NaOH, dialyzed for 8 hours against 0.9 per cent Nacl at
4°C,and added to the experimental medium in amounts equivalent

to 3 hypothalami/m1.

The results of this experiment (Table II-b) show that,
under our experimental conditions, prolactin inhibiting
capacity of the acid extract of the hypothalamus was lost
following dialysis. This suggests that the prolactin in-
hibiting factor (PIF) of the hypothalamus is dialyzable, and
is probably a small molecule. This is in agreement with work
on the other hypothalamic factors (CRF, LRF, TRF, GRF) which
indicates that they are probably all polypeptides (3).

0. Effects of Acid Extracts of Hypothalami from

Different Species on Pituitary Prolactin
Release In Vitro

 

 

 

An acid extract of rat hypothalamus inhibited synthesis
and release of pituitary prolactin lE.X$EEB: as shown in the
proceeding paper. It was therefore of interest to determine
whether hypothalami from other Species could similarly inhibit
pituitary prolactin release ig‘vigpg.

Ly0philized powders of pig, sheep, and bovine hypotha-
lamic extracts were supplied by Dr. Steelman (Merck, Sharpe,
and Dohme, Rahway, N. J.). Hypothalami 0r cerebral cortical
tissues from these Species were removed as quickly as possible
and immediately frozen. The tissues were homogenized with 5 ml
of 0.1N Hcl/gm of brain tissue (wet weight), centrifuged
(12000 x G) for 30 minutes, at 4°C. The supernatant was
neutralized with NaOH and ly0philized. The ly0philized powders
were tested for prolactin inhibiting activity by the method

described in Experiment 3'in the preceding paper.

 

pie.

The results in Table II-c show that acid extracts of

sheep, or bovine hypothalami inhibited pituitary pro-

lactin release ip vitro. An acid extract of cerebral cortical

tissue was ineffective. This indicates that hypothalami from

these Species contain the prolactin inhibiting factor (PIF).

REFERENCES

Lederis, K. Gen. Comp. Endocrinol. l: 80, 1961.

 

Buday, P. v. Nature. 191: 245, 1961.
Advances In Neuroendocrinology, edited by A. V. Nalbandov,

University of Illinois Press, Urbana, 1963.

TABLE II-a.

Effect of Bradykinin on Pituitary Prolactin
Release Ip'Vitro

f

 

 

 

No. Total Prolactin:

Dose/m1 Pairs No. IU/lOO mg AP*

Medium of No. of of Control Experimental P
(2x) Flasks Assays Pigeons (C) (E) CXEE
50 pg 1: 2 16 1.4” 1.6 >o.2o

:0.2 :0.1

 

*AP = Anterior Pituitary

**Mean :_Standard error of mean

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III. _I_N_ V 0 AND pt VITRO PROLACTIN SECRETION
BY TRANSPLANTED RAT "MAMMOTROPIC"

P ITITU ARY TUMORS

SUMMARY

The capacity of the functional, tranSplanted, autono-
mous, rat pitituary "mammotropic" tumor (Furth, MtT.F4) to
elaborate prolactin £2.1iY2 and iplyitpp_was investigated.
Prolactin content of transplanted anterior pituitary (AP)
and MtT. F4 were 1.6% and 4.8%, reSpectively, of that
present in the AP $2.§$EE° Considerable amounts of prolactin
could be detected in the blood plasma of rats bearing MtT.F4,
the levels being higher in rats with larger than with smaller
tumors. Prolactin ctivity could not be detected by the
assay method employed, in the blood plasma of normal cycling,
estrogen-treated, pregnant or lactating rats or in rats
bearing a single AP-tranSplant underneath the kidney capsule.
The amount of prolactin released into the medium by MtT. F4
during 3 days of culture was 8-31 times greater than that
present initially, indicating active synthesis and release
of prolactin by the tumor. These results show that MtT. F4,
like an AP-tranSplant removed from hypothalamic inhibition,
synthesises prolactin and releases it rapidly, leaving little

in the tissue itself.

The pioneer work of Furth has led to the identifica-
.tion and isolation of variety of tranSplantable hormone-
secreting rat and mouse pituitary tumors (7). One of these
is a chrom0phobic, autonomous rat pitituary "mammotr0pic"
tumor, strain F4 (MtT. F4) originally induced by estrogen
(6, 9). Following tranSplantation in the rat this tumor
appears to elaborate prolactin, STH and ACTH, but no FSH-LH
or TSH (2, 3, 7, 9, 21).

Animal cell cultures rarely perform differntiated
functions characteristic of the tissue of origin; this has
been especially true of cultures of endocrine tissue. How-
ever, our laboratory (13, 14) as well as others (ll, 15)
have recently observed that cultures of rat anterior pitui-
tary (AP) actively secrete prolactin in the medium; whereas
elaboration of all other hormones is markedly diminished.
The purpose of this study was to determine the capacity of
MtT. F4 to secrete prolactin ip_yiyg and EB.XIEEE as com-
pared to that of normal rat AP. A report of the present

work has previously been published in abstract form (19).

MATERIALS AND METHODS

Animals. Mature, virgin female, highly inbred Fischerl

rats of the CDF strain, 3-4 months old, were used in this
study. The rats were maintained in a temperature controlled
(75 :_1°F) and artifically illuminated (l4 hr/day) room.

They were fed ad libitum on Wayne Lab Blox pellets supplemented

with canned (Dash) dog food. We have observed that White
King Squabs 5-8 weeks old are more sensive for prolactin
assays than White Carneau pigeons of the same age. There-

fore, the former birds were used for all assays.

Prolactin assay. The sensitive intradermal pigeon-
cr0p assay method was utilized. The test material was
injected over one side of the crop-sac, while standard NIH
prolactin was injected over the other side of the crop-sac
in the same pigeons. Prolactin activity was expressed as
IV/lOO mg of tissue (wet weight) or IU/lOO ml plasma. The

details of the assay procedure has been described elsewhere

(14).

TranSplantation and assays of MtT. F4 and AP. Serial

 

tranSplantation of MtT. F4 was carried out in our laboratory.
The tumor tranSplantations were made by injecting rats
subcutaneously in the back of the neck 0.1 ml of tumor mince
in an equal volume of medium 199 (pH7.4) containing strepto-
mycin (100 pg/ml) and penicillin G potassium (100 U/ml). In
some rats, injections were made at two sites, resulting in
the formation of two tumors. The tumors were palpable within
4-5 weeks. When the tumor reached 1-2 cm in diameter, it

was removed and a portion of it was used for cultures and

for histological examination; another part of it was weighed,
homogenized with physiological saline and assayed for pro-
lactin activity. A total of six tumors (passage 41) from six

rats were assayed separately for prolactin activity.

Intact rats were tranSplanted, underneath the kidney
capsule, with one AP from each of the donor rats of the
same strain and approximately of the same age group. Ten
days later the AP-tranSplants were removed, trimmed of
adhering tissue under a disecting micr0800pe, pooled from
10 rats each, weighed, homogenized with saline and assayed
for prolactin activity. Some of the AP-tranSplants were
used for histological examination. The AP from normal
cycling rats was also removed, homogenized with saline and

assayed for prolactin activity.

Collection of rat blood and assay for prolactin

 

activity. Rats were anesthetized with ether and blood was
collected in a heparinized syringe from the posterior
abdominal artery. After centrifugation, the plasma was'
dialyzed, 1yophilized and assayed for prolactin activity.
Rats in different states were used as blood donors, as

indicated in Table 2.

Cultures of MtT. F4. A modification of the watch

 

glass technique (5) was used, in which sterile, plastic
(3.5 cm x 1 cm) Petri dishes3 were employed. Platforms
were formed from stainless steel mesh rectangles (1 cm x
2.3 cm), bent at each end to make a platform 4 mm high.
These were placed in Petri dishes with 3 ml of culture
medium 199”, containing 2 U insulin,5 100 U penicillin

G potassium and 100 ug streptomycin per ml. For some of

the cultures, calf, rabbit or horse serum6; or calf serum
and chicken embryo extract7 were added to medium 199, as
shown in Table 3.

The MtT. F4 was removed and placed in a Petri dish
(10 cm x 1.5 cm) containing a few dr0ps of medium. The
tumor was cut into small pieces, each about 2-3 mm in
diameter, and two such explants were placed on a strip of
lens paper (1.5 cm x 3 cm), which then was placed on top
of the platform inside the Petri dish. The tumor explants
were cultured for 3 or 6 days at 35°C in 95% 02 - 5% 002
atmOSphere. A total 148 cultures using 6 MtT. F4 were
carried out. At the end of the culture period, medium
from 8-10 culture dishes was pooled, dialyzed, ly0philized
and assayed for prolactin activity. The explants were
fixed in Bouins fluid, sectioned at 6 u and stained with
hematoxylin and eosin. The details of the culture procedure
have been described elsewhere (14).

In order to determine whether prolactin activity could
be detected in medium without MtT. F4 cultures, medium 199
alone or medium 199 containing 20% calf, rabbit or horse
serum was incubated for 3 days. At termination, the medium
was dialyzed, ly0philized and assayed for prolactin activity.
Also pregnant rat mammary gland or pigeon crop gland was
cultured for 3 days and the medium was similarly treated

and assayed for prolactin. In another experiment, standard

NIH ovine prolactin was added to medium 199 or medium 199

containing 20% calf serum, and incubated for 3 days to

ascertain the effects of incubation on prolactin activity.
RESULTS

The average prolactin activity (Table l) of MtT.F4 and
AP-tranSplants were found to be 0.06 and 4.0.02 IU/100 mg
tissue (wet weight), respectively, as compared to 1.2 IU/100
mg of fresh AP. This represents 4.8 and < 1.6 per cent, re-
spectively, of the content of prolactin present in fresh AP.

No prolactin activity was detected in the rat blood
plasma (Table 2) from (a) 3-4 months old mature female
cycling rats (b) female rats treated daily with 10 pg
estradiol for 10 days (0) 11-12 day pregnant rats (d) 10 day
postpartum, lactating rats, and (e) rats with a single AP-
tranSplant of 10 days duration. However, considerable
amounts of prolactin activity could be detected in the blood
plasma of rats bearing MtT.F4. The plasma prolactin activity
was found to be higher in rats bearing larger as compared to
those with smaller tumors.

Explants of MtT.F4 actively secreted prolactin in
the culture media (Table 3). The amounts of prolactin
secreted during 3 days of culture (0.5-1.9 IU/100 mg MtT.F4)
was found to be 8—31 times more than the initial content of
MtT.F4 (0.06 IU/lOO mg MtT.F4). Prolactin was also detected
in the medium of monolayer cultures of MtT.F4 for 22 days
(unpublished). Neither indubation of the medium alone for

three days without MtT.F4, nor medium from cultures of rat

mammary gland or pigeon crop gland showed prolactin activity.
Prolactin potency was not altered when standard NIH ovine
prolactin was incubated for 3 days in medium 199 with or
without 20% calf serum.

Histological examination of explants at the end of
the culture period showed variable degree of maintenance.
The best maintenance of explants, comparable to uncultured
MtT.F4, was obtained when 20% calf serum was added to
medium 199 (Fig. l and 2).~ A section from an AP-tranSplant
underneath the kidney capsule, 10 days after tranSplantation,
is shown in Figure 3. These AP-tranSplants were viable and
were actively secreting prolactin, as indicated by mammary

growth of the hosts rats.
DISCUSSION

The prolactin content of MtT.F4 and AP-tranSplants is
very low as compared with that in the AP 1p.§itg. The MtT.F4
and AP-tranSplant secrete considerable amounts of prolactin
lg 1123, as indicated by mammary gland stimulation or blood
assays in rats with tumors. Therefore, the MtT.F4 and AP-
tranSplant have less capacity to retain (or store) prolactin

than normal AP ip_situ.

 

Placement of hypothalamic lesions, transection of the
pituitary stalk, transplantation of the AP to noncranial
sites, administrations of certain depressant drugs and

cultures of AP 13 vitro have all demonstrated that the

hypothalamus exert an inhibitary effect on prolactin secre-
tion (13). We have recently provided evidence suggesting
that the hypothalamus contains a factor(s) which inhibits
prolactin secretion $2.XEEEB (20). The decrease in the
capacity of MtT.F4 or AP-tranSplant to retain prolactin may
be due to removal of hypothalamic inhibition. This may
.result in impairment of the intracellular storange mechanism,
or increased permeability of the plasma membrane of the cells
of the tumor and AP-tranSplant.

Considerable amounts of prolactin could be detected
in the blood plasma of rats with a MtT.F4. The high blood
prolactin levels may be due in part to the large size of
the tumor (500-1000 times larger than a normal rat AP), and
it may actually secrete a smaller amount of prolactin per
unit weight. The MtT.F4 during 3 days of culture secreted
0.5-1.9 IU of prolactin per 100 mg MtT.F4, while under
similar conditions bovine and rat AP secreted 8-12 IU (Tal-
walker and Meites, unpublished) and 10-40 IU per 100 mg tissue
respectively (l3, 14). This would indicate that on a per
unit weight basis, the ip_yit£g capacity of MtT.F4 to secrete
prolactin is much lower than the normal AP. Nevertheless,
the MtT.F4 during 3 days of culture secreted 8-31 times
more prolactin than was present initially, indicating con-
tinuous synthesis and release of prolactin. It appears that
the low ip_yitpg prolactin secretion by MtT.F4 as compared

with normal AP cannot be attributed to increased inactivation

of the harmone, since incubation ES£.§S of the medium con-
taining prolactin did not alter its activity. Prolactin
was not detectable by the assay procedure used, in blood
plasma from normal cycling, estrogen-treated, pregnant or
lactating rats, or in rats with a Single AP-tranSplant.
However, utilizing the luteotrOpic activity of prolactin
as an assay, Wolthuis (22,23) could detect prolactin in the
blood plasma of rats under similar conditions.

The MtT.F4 was initially induced by Furth with
estrogen (6, 9). It has been observed that the immediate
circulatory connection with the hypothalamus is not necessary

for lg vivo proliferative (6), or for an ip vivo and ip_vitro

 

(l3, l6) prolactin secretary reSponse to estrogen. Estrogen
administration EE.XEXB has been found to deplete the rat
hypothalamus of prolactin inhibiting activity (Rather and
Meites, unpublished). Thus estrogen stimulation may lead
to pituitary tumor formation and increased prolactin secre-
tion, shnilar to MtT.F4, by a direct action on the AP or
hypothalamus. It should be noted that transplantation alone
of the AP to noncranial sites in certain strains of rats (6,
10, 12) and mice (1, 4, 8) leads to pituitary tumor formation.
Thus removal of hypothalamic inhibition to the AP, by various
procedures, may lead to increased prolactin secretion and
formation of prolactin secreting tumors.

In conclusion, the present study shows that the MtT.F4

synthesises and releases considerable amounts of prolactin lg

vivo and ip vitro although little protactin is present in
the tumor tissue itself. In these aSpects its behaviour
is analogous to the AP when removed from hypothalamic inhi-

bition.

10

REFERENCES

Bardin, C. W. and Leibeit, A. D. Ne0plastic changes
occurring in Pituitary Isografts in mice. Proc. Am.
Assoc. Cancer Research, 3: 93, 1960.

Bates, R. W., Clifton, K. H., and Anderson, E.
Prolactin and Thyrotr0phin Content of Functional
Transplantable Pituitary Tumors. Proc. Soc. EXpl.
Biol. and Med., 93: 525-26, 1956.

Bates, R. W., Milkovic, S., and Garrison, M. M. Con-
centration of Prolactin, Growth Harmone and ACTH in
Blood and Tumor of Rats with TranSplantable MammotrOpic
Pituitary Tumors. Endocrinology, 11; 943-48, 1962.
Boot, L. M., MUhlobock, 0., decke, G., and Tengbergen,
W. E. Further Investigations on Induction of Mammary
Cancer in Mice by Isografts of Hypohyseal Tissue.
Cancer Research, 22; 713-27, 1962.

Chen, J. M. The cultivation in Fluid Medium of Organ-
ized Liver, Pancreas and Other Tissues of Foetal Rats.
Exper. Cell Research, 1; 518-29, 1954.

Clifton, K. H., and Furth, J. Changes in Hormone
Sensitivity of Pituitary Mammotropes During Progression
from Normal to Autonomous. Cancer Research, 21: 913—20,
1961.

Furth, J. Vistas in the EtiOlogy and Pathogenesis of

Tumors. Fed. Proc., g9 865—73, 1961.

10.

ll.

12.

13.

l4.

15.

11

Gardner, W. U. Tumors in TranSplanted Pituitary Glands
in Mice. Am. Assoc. Cancer Research, 3: 113, 1960.

Kim, U., Furth, J., and Yann0poulas, K. Observations

on Hormonal Control of Mammary Cancer. I. Estrogen and
Mammotr0pes. Cancer Research, 31: 233, 1963.

Kullander, S. On Tumor Formation in Gonadal and
Hyp0physeal TranSplants into Anterior Eye Chambers of
Gonadectomized Rats. Cancer Research, 20: 1079-82, 1960.
Kullander, S. Studies on the Growth and Hormone Pro-
duction of Rat Hyp0physis in Tissue Culture. Acta
Endscrinologica, 43; 147-54, 1963.

Martins, T. Action des hautes doses d’ estrine sur
L'hyp0physe ip_§itp, ou greffée la chambre anteriere

de 1'0eil du rat. Compt. Rend. Soc. deBiol., 123: 702-4,
1936.

Meites, J., Nicoll, C. S., and Talwalker, P. K. The
Central Nervous System and the Secretion and Release

of Prolactin. Ip_A. V. Nalbandov (ed.) Advances In
Neuroendocrinology, University of Illinois Press, Urbana,
Chapt. 8, 1963.

Nicoll, C. S. and Meites, J. Prolactin Secretion In
Vitrp; Effects of Thyroid Hormones and Insulin. Endocrin-
ology, 72: 544-51, 1963.

Pasteels, J. L. Secretion of Prolactin by the Pituitary
in Tissue Culture. Compt. Rend. des seances de 1'Academie

des Sciences, 253: 2140-42, 1961.

l6.

l7.

l8.

19.

20.

21.

22.

12

Ratner, A., Talwalker, P. K., and Meites, J. Effect of
Estrogen Administrations Ip'ylyp_on Prolactin Release
by Rat Pituitary Ip_Vlt£2, Proc. Soc. Expl. Biol. and
Med., gig: 12-15, 1963.

Takemoto, H., Yokoro, K., Furth, J., and Cohen, A. I.
Adrenotropic Activity of Mammo-Somatotropic Tumors in
Rats and Mice. Cancer Research, 22; 917-24, 1962.
Talwalker, P. K., and Meites, J. Mammary Lobulo-
Alveolar Growth Induced by Anterior Pituitary Hormones
in Adreno-Ovariectomized and Adreno-Ovariectomized-
Hyp0physectomized Rats. Proc. Soc. Expl. Biol. and Med.
107; 880-83, 1961.

Talwalker, P. K., Ratner, A., and Meites, J. 1p Vivo

 

and Ip_yit£p_Prolactin Production by Rat ”Mammotropic"
Pituitary Tumors. Fed. Proc., 21: 196, 1962.
Talwalker, P. K., Ratner, A., and Meites, J. Ip,Vit£g_
Inhibition of Pituitary Prolactin Systhesis and Release
by Hypothalamic Extract. Am. J. Physiol., 295; 213-18,
1963.

Wherry, F. E., Trigg, L. N., Grindeland, R. E., and

.Anderson, E. Identification of the Hormones Secreted

by an Autonomous Mammotropic Pituitary Tumor in Rats.
Proc. Soc. Expl. Biol. and Med., 119; 362-65, 1962.
Wolthuis, 0. L. The effects of Sex Steroids on the
Prolactin Content of Hyp0physes and Serum in Rats.

Acta Endocrinologica.43:137-46, 1963.

23.

.Wolthuis, O. J., and deJongh, S. E. The Prolactin Pro-

duction of a Pituitary Graft and of the Hyp0physis lg
Situ. Acta Endocrinologica, 43: 271-79, 1963.

13

FOOTNOTES

* Published with the approval of the Director of the Michi-

. gan Agricultural Experiment Station as journal article
N0.

+ This investigation was supported in part by NIH grant
AM4784-04, the Michigan Cancer Foundation, and an

Institutional Research grant from American Cancer Society.

. Charles River Breeding Laboratories, Brookline, Mass.
. Nutritional Biochemicals Corporation, Cleveland, Ohio.

. Falcon Plastics, Los Angeles, California.

1
2
3
4. Difco Laboratories, Detroit, Michigan.
5. Eli Lilly and Co., Indianapolis, Indiana.

6 Microbiological Associates, Bethesda, Maryland.
7

. Cappel Laboratories, West Chester, Pennsylvania.

ACKNOWLEDGMENT

We express our sincere appreciation to Dr. Jacob Furth
and Dr. Untae Kim of the Department of Pathology, College of
Physicians and Surgeons, Columbia University, New York, New
York, for providing us with "mammotr0pic" pituitary tumors;
to the Endocrinology Study Section, NIH, for ovine prolactin;

and to T. E. Staley for technical assistance.

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Fig. l. A section frem transplanted "mammotr0pic"
pituitary tumor, strain F4 (MtT.F4).

 

 

 

 

 

Fig. 2. A section from ”mammotr0pic" pituitary tumor
explant at the end of 3 days of culture in
medium ”199" containing 20%.calf serum.

 

Fig. 3. A section from ”mammotr0pic" pituitary
tumor explant at the end of 3 days of
culture in medium 199 without serum.

Fig. 4. An anterior pituitary tranSplant underneath
the kidney-capsule 10 days after tranSplan-
tation.

 

 

 

III. ADDENDUM

A. Prolactin Assays of Different Pituitary
"MammotropiC“ Tumor Strains

 

 

Prolactin assays of different pituitary "mammotr0pic"
tumor (MtT.) strains of rats and mice were carried out.
These tumors were supplied by Dr. Jacob Furth in the form
of frozen or ly0philized material. All these tumors were
tranSplantable autonomous and monomorphous. F4 and F6 were
originally induced by eotrogen; W5 by xeradiation, and MtT/SO
x—radiation followed by estrogen (l). Tumors were assayed
according to the method already described. The results of
the assay are summarized in Table III-a. For comparison
similar assays of anterior pituitaries from CFN and Fischer
strains of rats are also included. These data show that
prolactin content of these tumors is uniformly very low as
compared to normal rat AP in EEEE: with the exception of
mouse MtT/SO which has about half as much prolactin activity
as normal rat AP.

B. TranSplantation of Pitituary "Mammotropic"Tumor

Strain Fa (MtT.Fuf’in Sprague Dawley Rat

MtT.F4 was originally induced by diethylstilbestrol
treatment in inbred Fischer rat. The tumor was initially
dependent, in that following tranSplantation it grew only
in estrogen treated rats. After 4-5 serial passages in the
Fischer rat, it became autonomous, and could grow in normal

hosts following tranSplantation. However, it still retained

its reSponsiveness to estrogen, as indicated by increased
growth (2). Earlier attempts to tranSplant MtT.F4 in other
strains of rats were uniformly unsuccessful. Since the MtT.F4
which was available to us has passed through about 40 serial
passages in the Fischer rat, it was decided to determine
whether by this time it could be tranSplanted successfully

in the Sprague-Dawley strain of rats. The selection of the
Sprague-Dawley strain for this study was due to our interest
in mammary turmorogenesis. The mammary gland of the female
Sprague-Dawley rat is more susceptible to turmorogenesis
following intragastric (3, h) or intramammary administrations
(unpublished observations) of chemical carcinogens than other
strains of rats. Secondly, Sprague-Dawley rats are in no way
related to the Fischer strain (Dr. W. Dunning, personal com-
munication).

Sprague-Dawley female rats, 5-6 week old, were obtained
from Hormone Assay Laboratory, Chicago, Illinois. MtT.F4
(passage 40 and 41) was tranSplanted subcutaneously, into
animals 7-8 weeks old. The details of the transplantation
procedure has been described previously. Three weeks after
tranSplantation, rats were examined by palpation every week
for tumor develOpment. One group of rats was injected with
estrodiol in oil, 10 Pg, thrice weekly for six weeks, begin-
ning one day before tumor tranSplantation. Another group of
rats was treated with 50 IU PMS and 3 days later 25 IU HCG,
followed a day later by a tumor transplant. At termination

of the experiment all tumors were examined histologically.

The results are summarized in Table III—b, and show
that (a) successful tranSplantation in Sprague-Sawley rat
occurs only in 40-60% of the animals as compared with a
100% take in Fischer rats (b) estrogen or PMS and HCG treat-
ment had no effect in altering tumor incidence and (c) mean
lateney (7-8 weeks) was longer in Sprague-Dawley rats when
compared with Fischer rats (3-5 weeks). It was also observed
that the tumors in the Sprague-Dawley rats grew very slowly
and even after five months, they never attained more than
about 1/3 the size of tumors in Fischer rats. By this time
most of the rats started dying of respiratory infections.

An incidental observation was that most of these rats had
stomach ulcers. We have not observed this latter phenomenon
in Fischer rats.

Histological examination of these tumors showed that
they in no way differed in appearance from original tumor
tranSplant. Also the types of hormones secreted by this
tumor appeared to be similar to that of original tumor.
Enlarged adrenals and almost complete disappearance of thymus
indicated ACTH release. Increased in the sizes of liver,
kidney, and Spleen denoted somatotrOpic effects, while in-
tense mammary gland stimulation suggested prolactin secretion.
Similar effects are also observed when MtT.F4 is tranSplanted

in the Fischer rat (1, see Figures 1-4)-

REFERENCES

Takemoto, H., K. Yokoro, J. Furth, and A. I. Cohen.
Cancer Research, fig; 917, 1962.

Clifton, K. H. and J. Furth. Cancer Research, El: 913,
1961.

Huggins, C., G. Briziarelli, and H. Sutton. J. Exptl.
Med., $29: 25, 1959.

Harris, 0., H. Shay, M. Gruenstein, and J. L. Ciminera.

Acta Union Internatl. Cont. Cancer., $2} 595, 1959.

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Fig. l. "Mammotr0pic” pituitary tumor (MtT.F4)
in Fischer rat.

 

 

Fig. 2. "Mammotropic" pituitary tumor in Sprague-
Dawleywrat.

Fig. 3. Sprague—Dawley rat bearing "mammotr0pic"
pituitary tumor (MtT.F4) showing intense
mammary gland stimulation.

._..L—. ——___._‘ .i

 

 

 

 

Fig. 4.

Comparison of the different organs of (MtT.F4)
tumor bearing versus non tumor bearing Sprague-
Dawley rats. Note enlarged liver, kidney, and
adrenals of tumor bearing rat (left) as com-
pared with non tumor bearing rat (right).

 

 

awn}.

i:

an

 

SectiOn Two

 

EFFECT OF ANTERIOR PITUITARY HORMONES
ON INDUCTION OF NORMAL AND NEOPLASTIC

MAMMARY GROWTH IN THE RAT

I. MAMMARY LOBULO—ALVEOLAR GROWTH IN
ADRENO-OVARIECTOMIZED RATS FOLLOWING
TRANSPLANTATION OF "MAMMOTROPIC"

PITUI TORY TUMOR

The ovarian hormones estrogen and progesterone have
been shown to induce mammary lobulo—alveolar (L-A) growth
in intact or ovariectomized rats, and to synergize with
prolactin and growth hormone (STH) in adreno-ovaricetomized-
hypophysectomized rats (1). Ovarian hormones themselves
little or no effect on mammary L-A.development in the rat
in the absence of the anterior pituitary (AP), whereas our
laboratory has recently reported that AP hormones (STH and
prolactin) alone induced mammary L-A growth in the absence
of the ovaries, adrenals and pituitary (2). This suggested
that the pituitary hormones, STH and prolactin, are of
primary importance for mammary L-A growth in the rat.

Recently Furth and his co-workers (3, 4) developed
many types of tranSplantable pituitary tumors. One of these
is a monomorphous pituitary ”mammotr0pic" tumor, strain F4
(MtT.F4), originally induced by estrogen (5). This tumor
secretes prolactin, STH and corticotrOpin. (ACTH) but no
FSH-EH or TSH in the host animal (6-10). The tumor provides
a continuous source of prolactin, STH and ACTH, which is
secreted in increasing amounts as the tumor grows in the
host. It was of interest, therefore, to determine the effect
of a MtT.F4 transplant on mammary growth in the rat in which
the sources of estrogens have been eliminated by removal of

the ovaries and adrenals.

METHODS

Highly inbred, virgin female Fischer rats of the CDF
strain (Charles River Breeding Laboratories, Brookline,
Mass.), 3 months old, were used in this experiment. The
animals were fed §g_libitum on Wayne Lab Blox pellets,
supplemented with canned dog food (Dash) and slices of
oranges. Rats in Groups 1 and 2 (Table l) were sham-operated,
while those in Groups 3 and 4 were adreno-ovariectomized.
Following each Operation, the rats were injected once sub-
cutaneously with 30,000 units of penicillin G. The com-
pleteness of adrenalectomy was assessed in 2 ways: (a) after
killing the rats at the end of experiment, they were carefully
examined with a magnifying lens for adrenal remnants; and (b)
9 rats of the same strain, age, and weight were adreno-
ovariectomized and given saline (1% Nacl) in their drinking
water for 7 days. From the 8th day onward, the rats were
given distilled water for drinking instead of saline. The
day on which each rat died, after saline withdrawal, was
recorded.

Seven days after the operation, rats in Groups 2 and A
were tranSplanted with a Furth pituitary "mammotr0pic" tumor
(MtT.F4, passage 42) subcutaneously in the dorsal neck
region. Each MtT.F4 from the donor rats was removed
asceptically, cut into pieces with iris scissors, and the
tumor mince was suspended in equal volume of medium 199 (pH

7.4, Difco Laboratories, Detroit, Mich.). This was injected

in a 0.1 m1 volume. The tumors attained palpable size within
4-5 weeks. Estrogenic activity in adreno-ovariectomized rats
was determined by vaginal smears.

Eight weeks after tumor tranSplantation, all rats in
Groups 1-4 were killed. Both inguinal mammary pads from each
rat was disected free, spread flat on cork and fixed in
Bouin's fluid. One mammary pad from each animal was stained
by a standard procedure (2) for gross examination, and rated

for development according to the following scale:

I = Few ducts; few or no end buds

II = Moderate duct growth; moderate number of end buds
III = Numerous ducts and branches; many end buds
IV = Numerous ducts and branches;moderate lobulo-

alvedar growth
V = Numerous ducts with many branches;dense lobulo—
alveolar growth, as in mid or lage pregnancy
The other mammary gland pad from the same animal was prepared
for histological examination, as described earlier (11).
Lactation ratings of 0 to 4 were assigned to each gland,
depending on amount of secretion observed microscopically (11).
The MtT.F4 from each of the tumor bearing rats was also removed

and weighed.
RESULTS

The data are summarized in Table I. The mammary glands

from intact controls (Group 1) consisted of moderately

developed ducts with few end buds (Fig. 1) and no secretion
(Fig. 2). MtT.F4 tranSplantation in intact rats (Group 2)
induced highly extensive mammary lobulo—alveolar growth

and intense mammary secretion (Rating :>>-4), far greater
than that observed during normal pregnancy and lactation.

Adreno-ovariectomy alone without an MtT.F4-tranSplant
(Group 3) resulted in marked regression of the mammary duct
and end bud system (Fig. 3). TranSplantation of an MtT.F4
in adreno-ovariectomized rats (Group 4) induced extensive
mammary duct growth and branching and moderate to extensive
L-A development (Fig. 4). These rats showed a variable but
small degree of mammary secretion (Fig. 5 and 6).

The average weight of the tumors from the adreno-
ovariectomized rats (9.9 gm) was significantly greater
(P<;0.01) than those removed from the intact rats (6.1 gm).
Vaginal smears of all adreno-ovariectomized rats did not
show any evidence of estrogenic activity. The 9 adreno-
ovariectomized rats (separate from those used in Groups 1-4)
died following withdrawal of saline and substitution with
distilled water, as follows: one rat on day 5, 2 on day 7,

2 on day 8, and one each on days 11, 12, 14, and 15.
DISCUSSION

In previous in vivo studies, using adreno-ovariecto-
mized—hypOphysectomized rats and mice (1), or in vitro

cultures of the mammary glands of mice (12), it was observed

that estrogen and progestrone were needed in addition to
prolactin and STH to induce mammary L-A growth. In the
present study, mammary L-A growth was induced by the
hormones secreted by MtT.F4. This is in agreement with
the similar findings by Clifton and Furth (13).

We have reported that the MtT.F4 secretes prolactin
.$2.1£X2 as well as in yitrg cultures (6), and other in yivg
studies (7-10) have demonstrated that it also secretes
STH and ACTH, but no FSH-LH or TSH. This indicates that
mammary L-A growth in adreno-ovariectomized rats bearing a
MtT.F4 was due to secretion of prolactin, STH and ACTH from
the tumor. However, our previous demonstration that only
prolactin and STH are needed to induce full mammary L~A
develOpment in adreno-ovariectomized rats (2), suggests
that prolactin and STH were primarily reSponsible for the
develOpment of mammary L-A system observed in the tumor
bearing rats. Lasfargues (14) also found that L-A growth
can be induced in mouse mammary gland cultures by addition
of STH or prolactin to the culture medium. Addition of
estrogen to the culture medium was not essential.

Ovariectomy and adrenalectomy appeared to be complete
in these rats. Vaginal smears of these rats did not show
any evidence of estrogen activity, indicating that~
estrogenic was not availble to synerize with prolactin and
STH to stimulate mammary L-A growth. At necrOSpy, gross

examination did not reveal the presence of adrenal ramnants.

The 9 adreno-ovariectomized control rats died within 15

days after saline withdrawal. However, most of the rats
showed a slight degree of mammary secretion, indicating

the possible presence of slight amounts of glueocorticoids.
The minimal requirement for initiation of mammary secretion
in the rat is prolactin and gluescorticoids (1). It is also
possible that the large amounts of prolactin secreted by

the tumor may have decreased the amount of a glueocorticoid

necessary to initiate mammary secretion.
SUMMARY

1. The influence of a hormone-secreting (prolactin,
STH and ACTH), autonomous, tranSplanted "mammotr0pic"
pituitary tumor (Furth, MtT.F4) on mammary lobulo-alveolar
(L-A) growth in adreno-ovariectomized(doubly operated)
Fischer rats was investigated.

2. The MtT.F4-tranSplant stimulated extensive mammary
L-A growth in intact or doubly-Operated rats. Intact tumor
bearing rats showed intense mammary secretion, while doubly
operated tumor bearing rats revealed only slight amounts of
mammary secretion. The average weight of MtT.F4 in doubly
Operated rats was higher than that in intact controls.

3. Completeness of adrenalectomy and ovariectomy was
checked. Nine control doubly operated rats failed to survive
more than 15 days after withdrawal of saline. Vaginal smears

of these rats did not reveal any estrogenic activity.

A. It is concluded that full mammary L-A growth can
be induced following adreno-ovariectomy by a MtT.F4—trans-

plant secreting prolactin and STH.

10.

ll.

12.

REFERENCES

Lyons, W. R., Li, C. H., Johnson, R. E. in Recent

Progress in Hormone Research (G. Pincus, Ed.), 1958,

 

V 14, 219, Academic Press, N. Y.
Talwalker, P. K., Meites, J. Proc. Soc. Exp. Biol.

 

and Med., 1961, V 107, 880.
Furth, J., Clifton, K. H., Gadsden, E. L., Buffet, R.
F., Cancer Research, 1956, V 16, 608.

 

Furth, J., Kim, U., Clifton, K. H. Nat. Cancer Inst.
Monograph, 1960, V 2, 148.

Clifton, K. H., Furth, J. Cancer Research, 1961, V 21,
913.

Talwalker, P. K., Ratner, A., Meites, J. Fed. Proc.,
1962, V 21, 196.

Bates, R. W., Milkovic, 8., Garrison, M. M. Endocrinology,
1962, V 71, 943.

Kim, U., Furth, J., Yann0poluos, K. J. Natl. Cancer
Inst,, 1963, V 31, 233.

Takemoto, H., Yokoro, K., Furth, J., Cohen, A. I. Cancer

Research,1962, V 22, 917.

 

Wherry, F. E., Trigg, L. N., Grindeland, R. E.,Anderson,
E. Proc. Soc. Exp. Biol. and Med., 1962, V 110, 362.

 

Meites, J., Nicoll, C. S., Talwalker, P. K., Proc. Soc.

 

Exp. Biol. and Med., 1959, V 101, 563.
Elias, J. J., Rivera, E. Cancer Research, 1959, V 19, 505.

13. Clifton, K. H., Furth, J. Endocrinology, 1960, V 66, 893.

 

14. Lasfargues, E. Y., Murray, M. R. Developmental Biol.,

 

1959, V l, 413.

FOOTNOTES

* Published with the approval of the Director of the
Michigan Agricultural Experiment Station as journal
article No. .

 

** This investigation was supported by grants from NIH
(AM 4784-04),the Michigan Cancer Foundation, and an
Institutional Research grant from American Cancer

Society.

ACKNOWLEDGMENT

We express our sincere appreciation to Dr. Jacob
Furth and Dr. Untae Kim of the Department of Pathology,
College of Physicians and Surgeons, Columbia University, New
York, N. Y., for supplying "Mammotropic" pituitary tumors;

and T. E. Staley for technical assistance.

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Fig. 1. Intact controls. Moderate duct develOpment
and few end buds.

a O to rflw avg !”W

.3
O
.O.

.r..ln
Life:

_o 16.1..

.73..."

E.

 

Fig. 2. Mammary gland section from the same animal
as in Fig. 1. No mammary secretion present.

 

Fig, 3. Adreno-ovariectomized rat without MtT.F4
transplant. Few ducts with little branching.

L,

 

Fig._4. Adreno—ovaricetomized rat with MtT.F4 tranSplant.
Extensive lobulo-alveolar growth.

 

Fig. 5. Same type of rat as in Fig. 4, showing slight
mammary secretion.

 

Fig. 6. Same type of rat as in Fig. 4, showing
,moderate mammary secretion.

 

II. INDUCTION OF MAMMARY LOBULO-ALVEOLAR GROWTH
BY ANTERIOR PITUITARY HORMONES IN ADRENO-
OVARIECTOMIZED AND ADRENO-OVARIECTOMIZED-

HYPOPHYSECTOMIZED RATS

Studies in ovariectomized-hypOphysectomized or in
adreno-ovariectomized-hyp0physectomized rats have indicated
that hormones from the anterior pituitary and ovaries are
both essential for inducing mammary lobulo-alveolar growth
(-3). The adrenals are believed to be of secondary impor-
tance, although it is recognized that they secrete several
steroids which can influence mammary growth (1-3). Only a
small degree of lobulo-alveolar develOpment has been produced
by injecting anterior pituitary extracts into ovariectomized
or adreno-ovariectomized rats (4). Recently, Clifton and
Furth (5) observed complete lobulo-alveolar develOpment in
adreno-gonadectomized male rats of the Fischer strain after
implanting "mammotr0pic" pituitary tumors intramuscularly.
No data were given as to completeness of adrenalectomy in
these rats. ,Since these pituitary tumors appear to secret
both STH and prolactin (5), it was of interest to determine
whether frequent injections of large doses of these two
hormones or of whole anterior pituitary powder could induce
lobulo—alveolar mammary growth in adreno-ovariectomized or

adreno—ovariectomized-hyp0physectomized rats.
METHODS

Mature virgin female Carworth rats of the CFN strain,
weighing 180-240 g each, were used in this study. Rats in
groups 1-5 (Table 1) were adreno-ovariectomized (doubly

operated) while rats in groups 6 and 7 were hypOphysectomized

by the parapharyngeal approach and 3 days later were adreno-
ovariectomized (triply Operated). Following each operation,
the rats were injected once subcutaneously with 30,000 units
of penicillin G. Completeness of adrenalectomy was assessed
in two ways: (a) after killing the rats at the end of the
experiment, they were carefully examined with a magnifying
lens for adrenal remnants; and (b) 17 rats of the same strain,
age and weight were adreno-ovariectomized and supplied with
tap water instead of 1% saline. Death occurred in 16 rats
on the following days: one rat on day 7, two on day 9, one
on day 10, three on day 11, three on day 12, two on day 13,
and one each on days 14, 15, 17 and 19. The remaining rat
was killed on the 24th day, and upon examination one adrenal
was found which had not been completely removed. The sella
turcica of all hypOphysectomized rats was examined for pitui-
tary fragments at the end of the eXperiment. Only rats with
complete pituitary removal are included in the results.
Twenty-four hours after adreno-ovariectomy, all groups
of rats were treated for 10 days as shown in Table l.
Hormones were injected thrice daily at intervals of approxi-
mately six hours between 9 A.M. and 9 P.M. Bovine STH* and
ovine prolactin* were dissolved in distilled water and anter-

ior pituitary powder+ was suspended in physiological saline.

 

*Donated by Endrocrinology Study section, NIH.

+Supplied through courtesy of Dr. J. D. Fisher of Armour
Pharmaceutical Company, Kankakee, Illinois.

All rats were fed ad libitum on Wayne Lab—Blox pellets sup-
plemented with canned Armour's Dash dog food, and were
given 1% saline to drink. Slices of oranges and 5% glucose
in their drinking water were made available to all hypophy-
sectomized rats. Body weights of all rats were recorded at
the beginning and end of each treatment.

The rats were killed on the day following the last
injection, and both inguinal mammary pads were dissected
free, Spread flat on the cork and fixed in Bouin's fluid.
One mammary pad from each animal was stained by a standard
procedure (6), and rated for develOpment according to the

following scale:

I = Few ducts; few or no end buds
II = Moderate duct growth; moderate number of end
buds
111 = Numerous ducts and branches; many end buds
IV = Numerous ducts and branches; with moderate

lobulo-alveolar (L-A) growth
V = Numerous ducts and branches with dense L-A

growth, as in mid or late pregnancy.
RESULTS

The data are summarized in Table 1. In doubly oper-
ated rats, mammary glands from saline treated controls
(Group 1) consisted of moderately developed ducts with few

end buds (Fig. 1. See published article in Appendix for

figures.) Treatment with STH (Group 2) induced extensive
duct growth with many branches and end buds but no L-A
growth (Fig. 2). Prolactin (Group 3) also induced duct
growth with many branches and end buds (Fig. 3), but 3 out
of 7 rats showed limited L-A develOpment. Combined treat-
ment with STH and prolactin (Group 4) induced extensive
duct growth and dense L-A develOpment comparable to that
seen in rats after mid or late pregnancy (Fig. 4). The
results with anterior pituitary powder (Group 5) were vari-
able and mammary growth was not equal to that produced by
combined STH and prolactin treatment, but the majority of
these rats showed some L-A develOpment. In the triply Oper-
ated rats, mammary glands from control animals (Group 6)
consisted essentially of few small ducts with little branch-
ing and few or no end buds (Fig. 5). Combined treatment
with STH and prolactin (Group 7) produced extensive duct
growth with many branches and moderate L-A develOpment (Fig.6).
Doubly or triply operated controls gained very little
in average body weight during the treatment, while rats
given STH gained an average of 43 g. Prolactin and anterior
pituitary powder produced average body weight gains of 14 g

and 20 g, respectively.
DISCUSSION

In previous studies with triply operated rats adminis-

tration of estrogen and progesterone or adrenal corticoids

were needed in addition to STH and prolactin to induce lobulo-
alveolar (L-A) growth (2). The results of the present experi-
ment show that in doubly or triply operated rate combined
treatment with bovine STH and ovine prolactin, or bovine
anterior pituitary powder alone, can induce L-A development.
Our results are therefore in agreement with those of Clifton
and Furth (5) who found good L—A develOpment in adreno—
gonodectomized male rats implanted with "mammotr0pic" pituitary
tumors. The main differences between our experiment and
those reported by earlier investigators are that we injected
prolactin and STH or whole anterior pituitary powder thrice
instead of only once daily; injections were begun the day
after surgery; and relatively large doses of thses hormones
were used. Also, our rats were mature females, three to four
months old, whereas Lyons §t_§1, (2) employed mainly young
rats of the Long-Evans strain.

Ovariectomy and adrenalectomy appeared to be complete
in these rats. The 16 adrenalectomized rats not given saline
died within 19 days. Since injections were begun on the day
after removing the ovaries and adrenals, the possibility can-
not be excluded that small amounts of steroid hormones were
present during the first few days of treatment. It is doubtful
however, that such small amounts if present would have been
sufficient to synergize with the anterior pituitary hormones
to induce full L-A growth. Clifton and Furth (5) implanted
their pituitary tumors in rats 4 to 16 days after adreno-

gonadectomy and still obtained good L—A develOpment.

The present results cannot be interpreted to mean that
ovarian and probably adrenal cortical hormones have no role
in normal mammary develOpment in rats. Estrogen and proges-
terone have been shown to induce full mammary growth in
intact or ovariectomized rats (1) and to synergize with
prolactin and STH in triply operated rats (2). However, our
results suggest that the anterior pituitary is of primary
importance in mammary develOpment in the rat, since anterior
pituitary hormones alone induced full mammary develOpment in
the absence of ovaries and adrenals, whereas ovarian or
adrenal cortical hormones have been shown to have little or
no effect on the mammary growth in the absence of the anter—

ior pituitary (1-3).

REFERENCES

l. Turner, C. W. in Sex and Internal Secretions (E. Allen,

 

Ed.), 1939, Williams and Wilkins, Baltimore, Md.
2. Lyons, W. R., Li, C. H., Johnson, R. E. in Recent

Progress in Hormone Research (G. Pincus, Ed.), 1958,

 

V 14, 219, Academic Press, N. Y.

3. Meites, J. Chap. 16 in Reproduction in Domestic Animals
(H. H. Cole and P. T. Cupps, Eds.), 1959, Academic
Press, N. Y.

4. Cowie, A. T., Folley, S. J. Endocrinology, 1947, V 40,

 

274.
5. Clifton, K. H., Furth, J. Endocrinology, 1960, V 66,
893.

6. Nandi, S. Hormonal Control of Mammogenesis and Lacto-

 

genesis in the C3H/He Crgl Mouse (University of Calif-

 

ornia publications in Zoology), 1959, V 65, 4, University

of California Press, Berkeley.

V. ADDENDUM

Clifton and Furth (5) reported minimal mammary secretion
in adreno-gonadectomized Fischer rats bearing mammotrOpic
pituitary tumors, suggesting that accessory adrenal cortical
tissues may be present in these rats. Histological examina-
tion of stained mammary sections from our adreno-ovariectomized
rats treated with prolactin and STH failed to reveal any

secretory activity.

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III. MAMMARY TUMOR INDUCTION BY ESTROGEN OR ANTERIOR
PITUITARY HORMONES IN OVERIECTOMIZED RATS
GIVEN 7, 12-DIMEHYL-1, 2-BENZANTHRACENE

It has been well established that ovarian and pituitary
hormones play a major role in the normal develOpment of
the mammary gland, and in the maintenance and growth estab-
lised mammary tumors in the rat (1-8). The pituitary
appears to be of primary importance in mammary growth in the
rat, since a combination of growth hormone (STH) and prolac-
tin (9) or tranSplantation of a pituitary ”mammotr0pic"
tumor (10, Talwalker, Meites and Dueben, unpublished), which
presumably secretes prolactin and STH (5, 11, 12), induced
mammary growth in adreno-gonadectomized rats. On the other
hand, ovarian or adrenal steriods have little or no effect
on mammary growth in the absence of the pituitary (1-3).
Induction of mammary growth by estrogen in intact rats
appears to be partially mediated through the stimulation of
pituitary prolactin and STH secretion (3,4).

The rapid induction of mammary carcinomas by intra—
gastric administration of 3—methylcholanthrene (3-Mc) or 7,
l2-dimethyl-1, 2-benzanthracene (DMBA) has been described
(13, 14). Hyp0physectomy prevents mammary tumor induction
by 3—Mc, whereas tumors can be induced in hypOphysectomized
rats provided estrogen, progesterone and STH are given
together with 3—Mc(15). Dao (16) reported that ovariectomy
before a single feeding of DMBA prevented mammary tumor
induction in the Sprague-Dawley female rat, but tumors
appeared when ovaries were tranSplanted concurrently or

within several days after DMBA administration. This suggests

that ovarian hormones are essential for mammary tumor induc-
tion by DMBA. Since ovarian hormones influence pituitary
prolactin and STH secretions, it was of interest to determine
the role of estrogen, or prolactin and STH, in mammary tumor
induction by single feeding of DMBA in ovariectomized

Sprague-Dawley rat.
METHODS

Sprague-Dawley, virgin female rats (Hormone Assay
Laboratories, Chicago, Illinois) were used in this experi-
ment. Rats were randomly divided into 6 groups. When they
reached 52 days of age, group 1 was sham-Operated, while
groups 2-6 were bilaterally ovariectomized. Seven days
later, all rats (groups 1-6) received, once only, 20 mg of
7, 12-dimethyl—1, 2—benzanthracene (DMBA, Eastman Organic
Chemicals, Rochester, N. Y.) in 1 ml corn oil, by intragastric
instillation. This dose of DMBA is considered Optimal, since
100 per cent of rats, so treated, deve10p mammary tumors (14).
Groups 1-5 were treated for 7 days before and 7 days after
(total 14 days) DMBA administration, as shown in Table 1.
Group 6 was treated, daily, with porcine STH and ovine pro-
lactin in increasing dosages, beginning a day after ovari-
ectomy and continuing as follows: Days 1-15, 1 mg each;
days 16-30, 1.5 mg each; days 31-45, 2 mg each; days 46-60,
2.5 mg each; and days 61-75, 3 mg each. All injections were

given subcutaneously. Estrodiol was injected in 0.1 m1 corn

oil; and porcine STH and ovine prolactin (l5 IU/mg) were
given in 0.2 m1 saline.

The rats were examined for tumors by palpation twice
weekly. Occurrence of the tumor was dated from the day of
feeding DMBA. The number and per cent of animals develOping
tumors (incidence) was determined at the end of 40 weeks,
and the range and mean time of appearance of first tumors
(latency) were calculated. Rats were killed when they
appeared ill, or when the tumors became so large that they
ulcerated and bled. At necrOpsy, the tumors were fixed in
10% neutral formalin, sectioned at 6 u, and stained with

hematoxylin and eosin.
RESULTS

Spontaneous mammary carcinomas in Sprague-Dawley rat
are of very rare occurrence (13, 14, 17). Mammary carcinomas
were the only type observed in these DMBA fed rats. No
fibrosarcomas were detected in the mammary region. The
mammary carcinomas were grossly nodular, white and soft.
Hemorrhage and central necrosis were often encountered,
especially when the tumors became large. The tumors contained
acini lined with many layers of epithelial cells (Fig. 1)
arranged to form gland—like structures with papillary pro-
jections in the lumina. The lumina of these glandular
structures were often filled with eosinophilic-staining

material. Metastases of these mammary tumors were not

observed, but often infiltration of adjacent muscles and
skin were found. The principal causes of death of rats ap-
peared to be due to large size of tumors develOping hemorrhage
and necrosis; and increased susceptibility to reSpiratory
infection.

The results are summarized in Table 1. When 20 mg
of DMBA was fed to the intact sham-Operated controls and
they were thereafter given saline (group 1), 100 per cent
of the animals developed mammary tumors within 52-110 (mean
68)days. Ovariectomy 7 days before DMBA administration com-
pletely prvented tumor induction (group 2). Limited treat-
ment of ovariectomized rats with 1 and 10 ug estradiol
(groups 3 and 4) increased mammary tumor incidence to 33
and 23 per cent, respectively. In the estrogen treated
groups, the mean latency was slightly higher and average
number of tumors per tumor bearing rat was lower than in the
sham-operated intact controls. Combined daily treatment
with STH and prolactin, either for 14 or 7t days (groups 5
and 6), increased mammary tumor incidence to 44 and 66 per
cent, reSpectively. In these rats also, the mean latency
was higher and the average number of tumors per tumor bearing

rat was lower than in the sham-Operated controls.
DISCUSSION

Recent investigations have led to the concept that
mammary carcinogenesis involves two separate phases (a) initia-

tion of carcinogenesis by a carcinogen and (b) promotion of

\J'l

tumor growth by hormones. Carcingoens bring about an ir-
reversible change of the mammary cells, whereas hormones
regulate growth and function of these cells. Hormones them-
selves apparently are not carcinogenic (4, 16). The results
of the present study show that estradiol, or porlactin and
STH, administered during the initiation phase of mammary
carinogenesis by DMBA, permit ovaricetomized rats to deve10p
mammary carcinomas. Thus neOplastic transformation in the
cells of the mammary gland of rats requires the participation
of hormones, particularly prolactin and STH. Estrogen may
participate in the initiating phase through stimulation of
prolactin and STH secretion by the pituitary, and also by a
direct action on the mammary gland.

It is evident from the present study that estrogen, or
prolactin and STH, were only partially effective in inducing
mammary tumors in ovariectomized rats fed DMBA. This may be
due to the particular doses of hormones used, which may not
have been Optimal, and to the schedule and duration of the
hormone treatments. It is also possible that other hormones
may be involved, including progesterone, TSH or ACTH. However,
our study clearly show that limited treatment with the pitui-
tary hormones, prolactin and STH, in the absence of the
ovaries, can induce mammary tumors with the aid of a single
DMBA feeding.

Mammary tumors induced by 3-Mc or DMBA feeding in rats,

are hormone dependant during the early stages of growth, and

hence they may regress when apprOpriate endocrine glands
supplying the hormones are removed. Thus, growth of mammary
tumors is inhibited by ovariectomy, ovariectomy and adrenal~
ectomy or by hypOphysectomy; and stimulated by prolactin

and STH secreting pituitary "mammotr0pic” tumor transplant
(4—8, l3, l4). Estrogen maintains or stimulates growth of
these mammary tumors only in ovariectomized or ovaricetomized—
adrenalectomized rats. It is ineffective in the absence of
the pituitary (18). Therefore, estrogen may promote growth
of these mammary tumors through stimulation of secretion

by the pituitary, prolactin and STH. The present study,
together with others (4-6, 15, 18) suggest that the pituitary
hormones, prolactin and STH, are of primary importance both
in the initiating and promoting phases of mammary carcino-

genesis by 3-Mc or DMBA feeding in the rat.
SUMMARY

1. The effect of estrogen or prolactin and STH on
mammary tumor induction by a single feeding of 20 mg of 7,
l2-dimethy1-l, 2-benzanthracene (DMBA), in ovariectomized
Sprague-Dawley rat was investigated.

2. After DMBA administration, mammary tumor incidence
in sham-Operated controls was 100 per cent. In ovariectomized
rats, DMBA failed to induce any mammary tumor by the end of
40 weeks. However, daily treatment with 1 or 10 pg estradiol,

for 7 days before and after (total 14 days) DMBA administration,

induced mammary tumors in 33 and 23 per cent of rats, reSpec-
tively. Similar treatment with 1 mg STH and 1 mg prolactin,
twice daily, for 14 days increased mammary tumor incidence to
44 per cent, while continuous daily treatment with increasing
amounts of prolactin and STH for 75 days, starting with 0.5
mg and rising to l, 1.5, 2, 2.5 and finally to 3 mg each per
rat, at intervals of 15 days, resulted in a 66 per cent
incidence of mammary tumors.

3. The average number of tumors per tumor bearing
rat was lower (1.0—2.3) and mean tumor latency was longer
(89-146 days) in ovariectomized rats than in sham-Operated
controls (3.9, and 68 days, respectively). All tumors were
mammary carcinomas.

4. It is suggested that estrogen, prolactin and STH
participate in the induction phase of mammary carcinogenesis
by DMBA, and that the effect of estrogen on mammary tumor
induction is partially mediated through stimulation of pitui-

tary prolactin and STH secretion.

10.

11.

12.

REFERENCES

Lyons, W. R., Li, C. H., Johnson, R. E. in Recent

Progress in Hormone Research (G. Pincus, Ed.), 1958,

 

V 14, p. 219, Academic Press, N. Y.

Nandi, S. Hormonal Control of Mammogenesis and

 

Lactogenesis in the C3H/He Crgl Mouse (Univ. of Calif—

 

ornia publication in Zoology), 1959, V 65, p. 4, Univ.
of California Press, Berkeley.

Meites, J., Nicoll, C. S., Talwalker, P. K. in Advances
in Neuroendocrinology (A. V. Nalbandov, Ed.), 1963,

 

Chapt. 8, p. 238, Univ. of Illinois Press, Urbana.
Furth, J. Fed. Proc., 1961, V 20, 865.

 

Kim, U., Furth, J., Yann0poulos, K. J. Nat. Cancer Inst.,
19633 V 31’ 233.
Kim, U., Furth, J. Proc. Soc. Exp. Biol. and Med., 1960,

 

 

V 103, 643.

Dao, T. L., Sunderland, H. J. Nat. Cancer Inst., 1959,

 

V 23, 567.
Dao, T. L., Greiner, M. J. ibid., 1961, V 27, 333.

Talwalker, P. K., Meites, J. Proc. Soc. Exp. Biol. and

 

ygg,, 1961, v 107, 880.

Clifton, K. H., Furth, J. Endocrinology, 1960, V 66, 893.

 

Talwalker, P. K., Ratner, A., Meites, J. Fed. Proc., 1962,

 

V 21, 196.

Bates, R. W., Milkovic, 8., Garrison, M. M. Endrocinology,

 

1962, V 71, 943.

l3.

14.

15.
16.

17.

18.

Huggins, C., Briziarelli, G., Sutton, H. J. Exp..Med.,
1959, V 109. 25.
Huggins, C., Grand, L. C., Brillantes, F. P. Nature,

 

1961, V 189, 204.
Young, S. ibid., 1961, V 190, 356.

Dao, T. L. Cancer Research, 1962, V 22, 973.

 

Thomson, S. W., Husbey, R. A., Fox, M. A., Davis, 0. L.,

Hunt, R. D. J. Nat. Cancer Inst., 1961, V 27, 1037.

 

Sterenthal, A., Dominguez, M., Weisman, 0., Pearson,

O. H., Cancer Research, 1963, V 23, 481.

 

**

FOOTNOTES

Published with the approval of the Director of the
Michigan Agricultural Experiment Station as journal

article No.

 

This investigation was supported in part by grants from
NIH (AM4784—04), the Michigan Cancer Foundation; and an

Institutional Research Grant from the American Cancer

Society.

NIH postdoctoral fellow. Present address: Department

of Animal Breeding, Nihon University, Tokyo, Japan.

ACKNOWLEDGMENT

We eXpress our appreciation to the Endocrinology

Study Section, NIH, for ovine prolactin; to E. R. Squibbs,

for porcine growth hormone; and to T. E. Staley for tech-

nical assistance.

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.xix.
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OHHOOOH
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Fig. l. A mammary carcinoma in an ovariectomized rat
induced by a single feeding of DMBA and injections
of prolactin and STH. Hematoxylin and Eosin.

 

I'll, 1ll‘

Fig. 2. Mammary tumor induced by prolactin and STH
in the ovariectomized Sprague-Dawley rat
fed DMBA.

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III. ADDENDUM

A. Induction of Tumors in Different Strains of Rats
by Intramammary Administrations of ChemicaI
Carcinogens

 

Variations in the susceptibility of the rat mammary
gland to oral administrations of chemical carcinogens are
quite common. Thus an Optimal dose of methylcholanthrene

‘or dimethyl-benzanthracene will induce mammary tumors in 100
per cent of rats of the Sprague-Dawley strain, while Wistar
and Fischer strains are quite resistant to tumor induction by
this procedure. Also, the amount of carcinogen required per
animal to induce such tumors is relatively high (1-5).

Application of chemical carinogens to the skin in the
mammary gland region of the mouse produces skin as well as
mammary tumors. However, the latent period is unusually long
and the mammary tumor incidence is very low (6). Similar
applications of chemical carcinogens in the rat invariably
produces skin tumors (7, 8). Tumors have also been evoked in
rats and mice by subcutaneous injection of chemical carcinogen
or implantation of pellets containing chemical carcinogen. In
these studies, the latent period for tumor induction was
usually found to be considerably prolonged, depending upon
potency and dosage of the chemical carcinogen used. In the
albino rat the average latent period have been reported to be
somewhere between 21-32 weeks (6-11). The present experiments

were intended to determine the susceptibility of the mammary

glands of different strains of rats to mammary tumors follow-
ing intrammary administrations of different chemical carcino-

gens.

METHODS

3-methylcholanthrene (Mo, 3, 4-beanyrene or 9, lO-di-
methyl-l, 2-benzanthracene (DMBA) were dissolved in corn oil
and injected into the mammary gland fat pad in 1 mg doses and
in a 0.1 m1 volume, twice weekly for 4 weeks. All injections
were made approximately at the same site. Six separate
experiments on a total of 352 animals were performed in four
different strains of female rats, each 45 to 50 days old at
the beginning of the experiment.

Vasectomized male rats were introduced in the cages of
one group of rats of the Sprague—Daney strain treated with
Mo. Vaginal smears of these rats showed pseudOpregnancy-like
cycles. Starting on the fifth week, each rat was palpated in
the inguinal region once weekly for the presence of a tumor.
When these tumors appeared, they were measured with calipers once

weekly, and were removed for histological study when they

reached 15-20 mm in diameter.
RESULTS AND CONCLUSION

The results of these experiments are summarized in Tables
VI-a—l to VI-a—6. It will be noted that the vast majority of
the tumors produced by the three carcinogens were sarcomas

classified under three different categories by Dr. Langham:

(a) Fibro (b) Rhabdomyo and (c) Leiomyo, according to the
similar system suggested by Bonser (6). Of these the fibro
sarcoma was the most common type.

The incidence of adenocarcinoma of the mammary gland
was much higher (33%) in the Sprague-Dawley strain than in
Chemistry Department (M.S.U.), Carworth CFN (originally
Wistar), or in the highly inbred (32nd generation) Hunt-
HOppert strain of rat (0-7%). In the Sprague—Dawley strain,
MC and DMBA were more effective in inducing mammary carcinomas
than BP. PseudOpregnancy induced by vasectomized male rats
increased mammary tumor incidence by 10% only (from 50 to 60%)
when MC was used as a carcinogen in the Sprague-Dawley strain.

Most of the tumors in all four strains of rats develOped
in a comparatively short period of time (9-14 weeks) after
initiating treatment with the carcinogens, and the average
tumor incidence was very high (70-100%). Only one tumor was
produced under each injection site.

It is concluded that among the 4 strains of rats studied,
the mammary gland of Sprague-Dawley strain iS the most suscep~
tible to production of mammary tumors following intramammary
injections of chemical carcinogens, Among the three carcinogens
studied, MC and DMBA were more effective than BP in producing
mammary tumors in the Sprague—Dawley strain. The high
susceptibility of the mammary gland of the Sprague-Dawley rat
to carcinogens is of considerable interest, since this strain
of rats deve10ps mammary tumors almost exclusively when given

carcinogens orally (1, 2; also see the preceeding paper).

10.

11.

REFERENCES

Huggins, C., Briziarelli, G., and Sutton, H. J. Exptl.

wig” 109: 25, 1959.

Huggins, 0., Grand, L. C., and Brillantes, F. P.

Nature, 189: 204, 1961.

Synder, K. L., Butenandt, 0., Brillantes, F. P., and
Huggins, C. J. Natl. Cancer Inst., 29: 805, 1962.

 

Harris, 0., Shay, H., Gruenstein, M., and Ciminera, J. L.
Acta Union Internatl. Cont. Cancer, 15: 596, 1959.

 

Shay, H., Gruenstein, M., and Kessler, W. B. J. Natl.

Cancer Inst., 27: 503, 1961.

 

Bonser, G. M. J. Path. and Bact., 68: 531, 1954.

 

Dunning, W. F., Curtis, M. R., and Friedgood, C. E.

Cancer Research, 8: 83, 1948.

 

Dunning, W. F., Curtis, M. R., and Wood, F. C. £Eh_g,
Cancer, 39: 70, 1940.

Holmgrin, H., and Woulfort, G. Cancer Research, 7: 686,
1947.

Eker, R. Acta Path. Microbiol. Scandinav., 22: l, 1945.

 

 

Wolgom, W. H. Am. J. Cancer, 40: 321, 1940.

 

Tumor Induction with Chemical Carcinogens in MSU Chemistry

Department Rats*

TABLE VI-a-l.

 

 

-oKmoIaq

 

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8
L:
CU
U)

-OJ9IH

ewouIOJeQ

fiaemmew

sxeoM

(aouaprouI %og)
Folded QUGQPT

paaeeddv
JOMHL qsaIg

uaqM_sxoaM 'oN

aouopIouI
JOMHL

saownI warm SOEH
JO 'ON quom

poaeaddv down;
QSJIH UGHM
sqeg JO 'oN

quemIaadxg
JO HquuIBeg
49 8198 JO 'ON

Treatment

 

12
24
12

Controls

21

10

23 96

11

24
12

MC

10

l2
14

91

BP

70

10

13

DMBA

34

Total

 

*See text for abbreviations and treatments.

Tumor Induction with Chemical Carcinogens in MSU Chemistry

Department Rats*

TABLE VI—a-2.

ofimoqu

 

£6
E
8 -ofiwopqeua
O
U)
-oana
emoutoaeo
Kaemwew
sxeem

(oouepIouI %og)
POTJSO QUBQET

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JOMUL qsatg
uaqm sxeam 'oN

%
eouepIouI
down;

saownm quM squ
JO 'ON quom

peaeaddv JOMUL
QSJIH USUM
saeu JO 'oN

quomraodxg
JO HquuIBag
39 8198 JO 'ON

 

Treatment

 

15
15
15
15

Controls

13
14

11
13
14

100

14
15
10

14
15
13

MC

100

BP

77

DMBA

 

*See text for abbreviations and treatments

Chemistry

Tumor Induction with Chemical Carcinogens in

Department Rats (Est. Treated)*

TABLE VI—a—3.

 

-ofimoteq

-0Amopqqu

Sarcoma

‘OJQTH

 

emoquaaQ
Aaewmaw

sxeom
(aouopIouI %og)
DOIJGJ QUGQBT

peaeeddv
JOMUL QSJTg
ueqM sxoem 'oN

aouepIouI
down;

saownm quM 8493
JO ”ON 1940;

paaeoddv down;

asara uaum
sqeg JO 'oN

quemIJedxg
30 Butuutfiag
19 SQEH J0 'ON

Treatment

 

12

Controls

10

10 10 100

10

MC

12

80

10

10

BP

12

71

DMBA

19

Total

 

* See text for abbreviations and dosages of treatments.

Tumor Induction with Chemical Carcinogens in Carworth (CFN) Rats*

.TABLE VI—a-4.

 

 

 

Sarcoma

-oAmoqu

-0Amopqaug

"OJQTH

 

emoutoaeg
Aaemwew

sxoam
(aoueptouI %og)
pOTJGJ QUSQBT

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Jomnm asaIg

uaqm sneam 'oN

%

eouapIouI
JOMUL

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J0 'ON IPQOL

paaeaddv JOMHL

asata ueuM
sqeg JO 'oN

quewIJedxg
JO BUIUUIBeg
39 SQEH JO 'ON

Treatment

 

15

Controls

11

100

15
15
11

15

MC

11

10

100

15
15

BP

12

84

13

DMBA

30

Total

 

* See text for abbreviations and treatments.

Tumor Induction with Chemical Carcinogens in Hunt-HOppert

Inbred Rats*

TABLE VI-a-5.

 

-o&moIaq

—0Kw0pqqu

Sarcoma

“OJQTH

 

BMOUTOJBQ
Aaemmew

sxeem
(aouepIouI %0§)
POTJGJ QUSQET

peaaeddv
JOMDL qsatg

uqu sxeaM ’ON

aouapIouI
JOMHL

saownm HqIM 8423
JO 'ON IBQOL

paaaeddv JOMHL
QSJTH USQM
sieg 50 'oN

quamIJadxg
30 BUIUUIBOE
39 saeg 30 'oN

Treatment

 

 

15

Controls

10

14
11

14 12 85
14

15

15
15
15

MC

12

93

BP

DMBA

22

Total

 

* See text for abbreviations and treatments.

Tumor Induction with Chemical Carinogens in Sprague-Dawley Rats*

TABLE VI—a—6.

 

-o£motaq

-o£wopqaqg

Sarcoma

-oana

 

amouroaeo
Kaemmew

sxaem
(eoueptoul %og)
poIJea queqeq

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down; asatg
ueqM sxeem 'oN

eouepIouI
Jownm

saownm HQTM squ
JO 'ON quom

paeeddv JOWUL

asaIa uaum
3493 30 'oN

quemtaadxg
jo Buxuutfieg
QR SQEH JO 'ON

Treatment

 

15
15
15
15

Controls

10
13
13

85

l2

14
14

MC

93

13

BP

45

11

DMBA

ll

IO 10 100

10

nancy and

PseudOpreg-
MC**

14

10

Total

 

* See text for abbreviations and treatments.

**Vasectomized males were introduced in the cages.

Fig.

1.

Mammary tumor develOpment in the Sprague-
Dawley rat following intramammary adminis-
trations of Mo.

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Fig. 2. Mammary carcinoma in the Sprague-Dawley
rat induced by intramammary injections
of Mo.

 

. :1
’1CC'.£ .U‘V

 

Fig. 3. Fibroadenoma induced in the Sprague-Dawley
rat following intramammary administrations
of Mc.

APPENDIX

Curriculum Vitae and list of papers
published during graduate studies at
Michigan State University in which
writer was an author of co-author.

CURRICULUM VITAE

NAME: Talwalker, Purnachandra Keshavrao

(First) (Middle)

DATE OF BIRTH: May 19, 1932

PLACE OF BIRTH: Indore, Madhya Pradesh, India

NATIONALITY: Indian SEX: Male

MARITAL STATUS: Single

PRESENT ADDRESS: Department of Physiology and Pharmacology

Michigan State University
East Lansing, Michigan, U.S.A.

PERMANENT ADDRESS IN INDIA: 963 A Krishnanagar,

Bhavnagar, Gujarat, India

EDUCATION QUALIFICATIONS:

Major Field of

 

 

Degree Year I Study
B.Sc.(Honors) 1953 Gujarat University, Chemistry
India

M.Sc. 1956 Gujarat University, Chemistry
India

M.S. 1959 Michigan State Univ. Physiology
East Lansing, Michigan
U.S.A.

Ph.D. 1964 Michigan State Univ. Physiology-Biochemistry
Endrocrinology as
special field

HONORS:

(a)

(b)

Awarded State Government of Sourashtra India
postgraduate fellowship for one year 1953-54)
for obtaining highest marks at the B. c. examination.

Elected as a full member of honorary national scientific
society,Sigma Xi, U.S.A.

POSITIONS HELD:

(a)

(b)

Teaching Assistant, Department of Biochemistry,
M.P. Shah Medical College, India, 1955—57.

Research Assistant, Department of Physiology and
Pharmacology, Michigan State University, U.S.A.,
1958-63: '

VITAE 2
Talwalker, Purnachandra Keshavrao

TALKS PRESENTED AT THE SCIENTIFIC MEETINGS:

Meeting Date Topic

1. 44th Ann. Meeting Fed. April 14, 1960 Initiation and main-
Am. Soc. Exptl. Biol., tenance of milk
Chicago, Illinois secretion following

chlorpromazine
administration.

2. 45th Ann. Meeting Fed. April, 1961 Initiation of mammary
Am. Soc. Exptl. Biol., secretion in pregnant
Atlantic City, New rats and rabbits by
Jersey hydrocortisone acetate.

3. 46th Ann. Meeting Fed. April 18, 1962 In_vivo and in vitro
Am. Soc. Exptl. Biol., prolactin prBduction
Atlantic City, New by rat "mammotropic"
Jersey pituitary tumors.

4. North Central Confer- May 6, 1962 In vitro evidence for
ence on Comparative prolactin inhibiting
Endocrinology, factor in the hypo-
Brooklodge, Michigan thalamus.

5. Ann. Meeting of Mich. March 26,1959 Effects of prolactin
Acad. of 80., East oxytocin and hydro-
Lansing, Michigan cortisone on lactational

performance of rats.

6. Ann. Meeting of Mich. March 27,1960 Effects of chlor-
Acad. of 80., promazine on mammary
Ann Arbor, Michigan glands of rats.

7. Ann. Meeting of Mich. March 24,1961 Induction of lactation
Acad. of SC., in pregnant animals by
Detroit, Michigan hydrocortisone acetate.

VITAE 3
Talwalker, Purnachandra Keshavrao

RESEARCH PUBLICATIONS

(During graduate studies at Michigan State University)

Co-author of a chapter in the book:

CNS and Secretion and Release of Prolactin. Chapter 8.
In: Advances in Neuroendocrinology, edited by A. V.
Nalbandov, University of Illinois Press, Urbana,
Illinois. 1963.

Articles:

1.

Talwalker, P. K., J. Meites, and C. S. Nicoll. Effects
of hydrocortisone, prolactin and oxytocin on lactational
performance of rats. Am. J. Physiol., 199: 1070, 1960.

Talwalker, P. K., J. Meites, C. S. Nicoll and T. F.
Hopkins. Effects of chlorpromazine on mammary glands
of rats. Am. J. Physiol., 199: 1073, 1960.

Talwalker, P. K. and J. Meites. Mammary lobulo-alveolar
growth induced by anterior pituitary hormones in adreno-
ovariectomized and adreno-ovariectomized-hyp0physectomized

(rats. Proc. Soc. Exp. Biol. Med., 107: 880, 1961.

Talwalker, P. K., C. S. Nicoll and J. Meites. Induction
of mammary secretion in pregnant rats and rabbits by
hydrocortisone acetate. Endocrinology, 69: 802, 1961.

Talwalker, P. K., A Ratner and J. Meites. In vitro
inhibition of pituitary prolactin synthesis and release
by hypothalamic extract. Am. J. Physiol., 205: 213, 1963.

Nicoll, C. 8., P. K. Talwalker and J. Meites. Initiation
of lactation in rats by nonspecific stresses. Am. J.
Physiol., 198: 1103, 1960.

Ratner, A., P. K. Talwalker and J. Meites. Effect of
estrogen administration in vivo on prolactin release in

vitro. Proc. Soc. Exp. BIOI. Med., 112: 12, 1963.

Meites, J., P. K. Talwalker and C. S. Nicoll. Failure of
oxytocin to initiate mammary secretion in rabbits or rats.
Proc. Soc. Exp. Biol. Med., 105: 467, 1961.

Meites, J., P. K. Talwalker and C. S. Nicoll. Initiation
of lactation in rats with hypothalamic or crebral tissue.
Proc. Soc. Exp. Biol. Med., 103: 298, 1960.

VITAE 4
Talwalker, Purnachandra Keshavrao

10.

ll.

12.

13.

14.

15.

16.

Meites, J., P. K. Talwalker and C. S. Nicoll. Induction
of mammary growth and lactation in rabbits with epine-
phrine, acetylcholine and serotonin. Proc. Soc. Exp.
Biol. Med., 104: 192, 1960.

Meites, J., C. S. Nicoll and P. K. Talwalker. Effects
of reserpine and serotonin on milk secretion and mammary
growth in the rat. Proc. Soc. Exp. Biol. Med., 101: 563,

1959.

Meites, J., C. S. Nicoll and P. K. Talwalker. Local
action of oxytocin on mammary glands of postpartum rats
after litter removal. Proc. Soc. Exp. Biol. Med., 103:
118, 1960.

Meites, J., C. S. Nicoll and P. K. Talwalker. Induction
and maintenance of lactation in rats by electrical
stimulation of uterin cervix. Proc. Soc. Exp. Biol.
Med., 102: 127, 1959.

Meites, J., T. F. HOpkins and P. K. Talwalker. Initiation
of lactation in pregnant rabbits with prolactin, cortisol
or both. Endrocinology, 73: 261, 1963.

Talwalker, P. K., A. Ratner and J. Meites. .In vivo and
in vitro prolactin production by rat "mammotr0pIc"
'pItuItary tumors. Federation Proc. 21: 196, 1962
(Abstract).

Mizuno, H., P. K. Talwalker and J. Meites. Inhibition
of mammary secretion in rats by Iproniazid. Proc. Soc.
Exp. Biol. Med. In press.

 

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