EFFECTS OF ADRENAL CORTICAL AID PITUITARY HORMONES ON
INITIATION AND MAINTENANCE OF LACTATION IN RATS

by
Robert Merrill Johnson

AN ABSTRACT
Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
Department of Physiology and Pharmacology
1957
Approved by

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Robert Merrill Johnson
1

ABSTRACT

1*

In Experiment I, physiological saline

(0*85 percent)

or 2 mg, doses of ACTH, cortisone or hydrocortisone were in­
jected daily for

days into ij.2 intact, mature female rats,

10

All three hormones produced growth of ducts,

y

lobule-alveolar

development, and secretory activity in the mammary glands.

This

was most marked for cortisone and least for ACTH at the dose
levels employed,
2,

Cortisone increased pituitary prolactin content by

about 23 percent, hydrocortisone by about Lpl percent, and ACTH
by about 71 percent,
3,

In Experiment II, 100 mature female rats were

ovariectomized and divided into ten groups of
according to weight.

rats each,

Eight groups were divided into two major

groups of L|_0 rats each (four groups of
maining two groups

10

(20

10

each) and the re­

rats each) were used for controls.

The treatment of the groups was as follows:
(1) controls, saline only,
(2 ) controls,

1

mg. cortisone only.

The two major groups received either 5 ug. or 10 ug. of
estrone.

Within these two major groups cortisone was administered

to each sub-group as follows:
daily, L}.)
J4..

mg. daily.

l) none,

2

)

1

mg. daily,

3

)

2

mg,

All injections were made for 10 days,

Cortisone alone stimulated lobule-alveolar develop­

ment and secretory activity in the mammary glands of the ovariectomized rats.

However, mammary growth was not as pronounced

as observed in the intact rats of Experiment I.

On the other

y:

Robert Merrill Johnson
2

hand, when cortisone and estrone were given together, marked
lobule-alveolar development was elicited and secretory activity
was greatly increased,
5•

Cortisone augmented the pituitary prolactin content

of ovariectomized-estrone treated rats at the

1

m g , level.

However, when I4. mg, of cortisone was given daily with

10

ug,

^

of estrone, there was an inhibition of estrone action on the
pituitary,
6

.

In Experiment III, the effects of cortisone on galac-

topoiesis was studied in 30 mature female rats.

These rats

were bred and at parturition their litters were reduced to
young each.

7

The dams were injected daily during an 18-day post­

partum period with 0.25# 0,5 or 1.0 mg. of cortisone.

The lac­

tational response was measured by the use of litter growth rate.
The rats receiving 0.5 mg* cortisone showed a significant in­
crease in milk yield during the peak (6 th- 1 0 th day post-parturn)
and during the declining phase of lactation (llth-l 8 th day
post-partum).
Cortisone at the 1.0 mg. level only slightly Increased the
average litter growth during the

18

-day experimental period.

However, during the declining phase of lactation,

significant

increases In litter weight gain were noted over that of the
controls.
6.
Experiment IV.

The results of Experiment III were confirmed in
In addition,

injections of cortisone were

continued for 10 days after the young were removed (l8 th- 2 8 th

Robert Merrill Johnson
3

day), In order to study its effects on mammary involution.
was found that cortisone at the
mammary involution.

1.0

It

mg. level markedly retarded

These mammary glands were comparable to

those of an untreated rat

5

days after removal of the young.

Cortisone at the 0.5 nig. level produced slightly less retarda­
tion of mammary disintegration,

comparable to that of an un­

treated rat six days after removal of its litter.
7.

In Experiment IV the effect of growth hormone,

prolactin, oxytocin ana ACTH on galactopoiesis and mammary
involution were studied.

Prolactin given at a dosage of 1 mg.

daily increased the average litter weight throughout the 18-day
post-partum period.

These increases were about equal to those

of the rats treated with 0.5 nig. of cortisone aaily.

When

cortisone, prolactin and growth hormone were administered to­
gether, the response was of about the same magnitude as cor­
tisone or prolactin alone.

Thus no synergistic action on galac­

topoiesis was exerted by these hormones.

Growth hormone, when

given alone, did not increase lactation.
8.

Prolactin (1 mg.) or prolactin, cortisone and

growth hormone given together, retarded mammary involution
comparable to that of a maraaary gland of an untreated rat
5 days after removal of the young.

Growth hormone alone at

the level employed did not retard mammary involution.
9.

Oxytocin and ACTH exhibited galactopoietic effects

in parturient rats comparable to those of prolactin or
cortisone-treated rats.

However, the former two hormones

Robert Merrill Joimson

showea no ability to retard mammary involution following re­
moval of the young for 10 days.
10*

In Experiment V, suckling decreased the pituitary

prolactin content in lactating rats.

Electrical stimulation

of the cervix of lactating rats appeared to increase pituitary
prolactin content over that of non-sucklea or suckleu rats.
Injections of oxytocin appeared to produce a large increase
in pituitary prolactin content over that of suckled, non-sucxled
or electrically stimulated rats.

It appears, therefore, that

neither oxytocin nor electrical stimulation of the cervix in­
duces a release of prolactin from the pituitary.

EFFECTS OF ADRENAL CORTICAL AMD PITUITARY HORMONES ON
INITIATION AND MAINTENANCE OF LACTATION IN RATS

by
Robert Merrill Johnson

A THESIS

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

DOCTOR OF PHILOSOPHY
Department of Physiology and Pharmacology
1957

Dedicated.
to
my wife

TABLE OF CONTENTS
Page
INTRODUCTION

1

REVIEW OF LITERATURE

k

Hormonal Control of Mammary Growth
1, Estrogens
2. Progesterone
3* Anterior Pituitary Hormones
Ij.* Adrenal Cortex
5« Involution of the Mammary Gland
during Lactation
Factors Controlling the Initiation and
Maintenance of Lactation
1* Role of the Anterior Pituitary
2. Adrenal Cortex
3* Suckling Reflex
Attempts to Increase Lactation (galactopoiesis ) or
Maintain Established Lactation with Hormones

k
5
6
8

10

$
13

17

EXPERIMENTAL
I.

II.

III.
IV.

V.

EFFECTS OF ACTH AND ADRENAL CORTICAL HORMONES
ON MAMMARI GROWTH AND PITHITART PROLACTIN
CONTENT IN INTACT RATS

20

EFFECT 01' ESTRONE AND CORTISONE ON PITUITARY
PROLACTIN CONTENT AND EAMMARI GROWTH OF
0 VARIECTOMIZED RATS

25

EFFECTS OF CORTISONE ON GALACTOPOIESIS IN T h e RAT

32

EFFECTS OF CORTISONE, GROWTH HORMQN e , PROLACTIN,
OXYTOCIN AND ACTH ON LACTATION AND INVOLUTION
OF MAMMARY GLANDS IN RATS

ko

FACTORS INFLUENCING THE RELEASE OF PROLACTIN
IN SUCKLING MOTHER RATS

58

DISCUSSION

62

SUMMARY

66

BIBLIOGRAPHY

72

APPENDIX

81

LIST OF TABLES
TABLE
1«

PAGE

Effects of cortisone, hydrocortisone and ACTH on
mammary growth and pituitary prolactin content
of rats

23

Effect of estrone ana cortisone on pituitary
prolactin content of rats

29

Effect of cortisone on body weights of dams and
litters, and on adrenal weights of rats

36

ip.

Effects of cortisone on litter weight gain in rats

37

5*

Effects of cortisone on "instantaneous growth rate"
of rat litters

38

Effects of cortisone, growth hormone, prolactin,
oxytocin and ACTH on body weight of rat mothers
and litters

u

2.
3«

6.

7*
8.

9*

Effects of cortisone, growth hormone, oxytocin,
prolactin and ACTH on body weights of rat litters

47

Effects of cortisone, growth hormone, prolactin,
oxytocin and ACTH on "instantaneous growth rate"
of litters

48

Release of prolactin in suckling mother rats

61

LIST OF FIGURES
FIGURE
1#
2.
3*

ip.
5*

Photomicrographs of mammary glands from rats receiving
saline, cortisone, hydrocortisone or ACTH

2lp

Graph showing the effects of estrone ana cortisone
on pituit ary prolactin content

30

Photomicrographs ofmammary glanas from rats receiving
saline, 10 ug, estrone, 1 mg. cortisone, or 10 ug,
estrone plus 1 mg. cortisone

31

Graph showing tne effects of cortisone
weight gains in rats

39

on litter

Graph showing the effects of cortisone,
growth
hormone, prolactin, oxytocin ana aCTH on litter
weight gains in rats

49

6.

Photomicrograph of a control mammary glana from a rat
10 days after young have been removed

50

7.

Photomicrograph of a mammary gland from
a rat
receiving 1 mg, cortisone daily for 10 days after
young were removed

51

Photomicrograph of a mammary gland from a rat
receiving 0 ,5 mg, cortisone daily for 10 days
young were removed

52

8.

9*

10.

after

Photomicrograph of a mammary gland of arat receiving
1 mg, growth hormone daily for 10 days after young
were removed

53

Photomicrograph of a mammary gland of arat receiving
1 mg. prolactin daily for 10 days after young were
removed

51f

11.

Photomicrograph
of a mammary gland of a rat receiving
0 . 5 mg. cortisone, 1 mg. prolactin, ana 1 mg. growth
hormone daily for 10 days after youngwere removed
55

12.

Photomicrograph of a mammary gland from
a rat
receiving 0.5 I* U. oxytocin twice daily for 10
days after young were removed

56

Photomicrograph of a mammary gland from
a rat
receiving 1 mg. ACTH daily for 10 days after
young were removed

57

13*

Figure
111 #

15.
16.
17•
18.
19*
20.
21*
22,
23*

Photomicrograph of
after removal of

Page
a control mammary gland immediately
young

83

Photomicrograph of a control marrmary gland from a rat
one day after removal of young

84

Photomicrograph of a control mammary gland from a rat
two days after removal of young

85

Photomicrograph of a control mammary gland from a rat
three days after removal of young

86

Photomicrograph of a control mammary gland from a rat
four days after removal of young

87

Photomicrograph of a control mairmary gland from a rat
five days after removal of young

88

Photomicrograph of a control mammary gland from a rat
six days after removal of young

89

Photomicrograph of a control mammary gland from a rat
seven days after removal of young

90

Photomicrograph of a control mammary gland from a rat
eight days after removal of young

91

Photomicrograph of a control mammary gland from a rat
nine days after removal of young

92

ACKNOWLEDGMENTS
The author wishes to express his sincere gratitude to
Dr. J. Meites, Professor, Department of Physiology and Pharma­
cology, for his generous assistance ana constructive criticism
\

throughout the course of this work ana during tne preparation
of the manuscript.

He also wisnes to express his appreciation

to Dr. B. V. Alfredson, head of tne Department of Physiology
and Pharmacology, for providing facilities a m
to carry on these experiments;

labpratory space

to Dr. E. P. Reineke whose assis­

tance and advice were most helpful during Dr. J. Meites'
sabbatical leave; and tc Dr. A. T. Hardy, H. D., Sparrow Hospi­
tal, for his assistance in the reading of the histological
sections.

The author also wishes to thank Dr. J. E. Nellor,

Dr. G. Hoppert, Dr, L. F. Wolterink and Dr. W. D. Collings,
for their advice during the course of this work.
Thanks are also due Dr. C. E. Black, M. D., Pathologist,
Sparrow Hospital, for the use of laboratory facilities, Miss V.
Perkins for her technical assistance, Mr. John Monroe for his
help in the care of experimental animals and Miss Joan Ahrenhold,
Mr. Gene Flamboe and Mr. Robert Collins who assisted in con­
ducting parts of the experiments.

Thanks are also due Dr. E.

Smith for her assistance in preparing the photomicrographs.
The author wishes to thank Dr. L. Michaud of Merck
and Company, Rahway, N, J., for the cortisone acetate and

hydrocortisone acetate, Dr. I. Bunding of Armour Laboratories,
Chicago, Illinois,

for ACTH and growth hormone, and Dr, A.

Borman of Squibb and Sons, New Brunswick, N. J,, for the pro­
lactin used In this work.
The writer is indebted to the Michigan Agricultural
Experiment Station, American Cancer Society and the Committee
for Research in Problems of Sex of the National Research Coun­
cil for providing financial support to Dr. J. Meites, which
enabled the author to carry out this work.
The author is particularly obligated to the Department
of Physiology and Pharmacology for providing a Graduate Assistantship for the three years of this work.

1

INTRODUCTION
It is widely accepted that mammary growth requires es­
trogen for duct growth, and progesterone and estrogen in com­
bination for lobule-alveolar development*

In some species,

I*e., the guinea pig, cow, goat, etc., estrogens alone in suf­
ficient amounts are capable of producing lobule-alveolar
growth.

A possible explanation is that the guinea pig and

other species may be able to secrete progesterone or other
adrenal cortical hormones from the adrenal cortex under estro­
gen stimulation, which may stimulate lobule-alveolar mammary
growth (Hohn, 1957).
Desoxycorticosterone

(DCA) has about one-third the mam­

mary growth activity of progesterone in mice (Mixner and Turner,
1914-3) and it is able also to stimulate mammary growth in guinea
pigs (Van Heuverswyn, ejt al, 1939; Nelson, 1937).
The importance of the adrenal cortical hormones for the
maintenance of lactation Is well established.

Adrenalectomy

during lactation results in cessation of lactation, but if the
adrenal cortical hormones are replaced, lactation can be main­
tained.

It has not been definitely established whether these

hormones are only necessary for the general maintenance of
/
carbohydrate, protein and electrolyte metabolism in the body
as a whole, or if they have a more direct role in the initia­
tion and maintenance of lactation.

2

It has been established that lactation in most mammals
rapidly reaches a peak and then slowly declines over a long
period of time*

Attempts have been made to increase milk

yield during lactation by the use of hormones.

Crude anterior

pituitary extracts given during the declining phase of lacta­
tion in cattle produces a temporary increase in milk secretion
(Asimov and Krouze,

1937; Folley and Young, 193^).

A more pro­

nounced increase can be obtained by the injection of growth
hormone while the galactopcietic effect of purified prolactin is
relatively small (Folley, 1955)*

This suggests that prolactin

is not the only hormone responsible for the galactopoietic
potency of the pituitary, and that other hormones of the
pituitary may act synergestically with prolactin.

Despite its

low galactopcietic potency, prolactin is essential for the ini­
tiation and maintenance of lactation.
Other factors leading to the decline in lactation are
the involutionary changes in the mammary gland (Selye and
McKeown, 193l+; Turner and Reineke, 193&)*

It is well established

that suckling or milking causes the release of pitocin and pro­
lactin which may be at least partially responsible for the main­
tenance of the mammary gland.

Williams (191-1-5) has shown that

prolactin is capable of retarding mammary involution in par­
turient, non-suckled rats.

However, little is known of the

action of other hormones on the maintenance of tne mammary
gland during lactation.

The adrenal cortex may play a role

3

since Gregoire

(191+7) and others have shown that ACTH is re­

leased during suckling.
This thesis is an attempt to provide additional in­
formation on the relation of the adrenal cortex ana pituitary
hormones on the initiation and maintenance of lactation.
specific problems studied were:

The

(l) the effects of adrenal

glucocorticoids and ACTH on mammary growth and secretion;
(2)

the effects of ACTH and glucocorticoids on pituitary pro­

lactin content;
cortisone,

(3 ) tne possible galactopoietic effects of

prolactin, oxytocin, ACTH and growth hormone in

lactating rats;

(1+) the effects of all these hormones on the

retardation of mammary involution after parturition, and
(f>) the effects of oxytocin on prolactin release in the lac­
tating rat.

b

REVIEW OP LITERATURE
Hormonal Control of Mammary Growth
Estrogens
It was concluded in earlier work that duct growth could
be induced with estrogen alone, but that complete development
of the lobule-alveolar systems required progesterone as well.
While estrogen stimulates duct growth in all species,

the

lobule-alveolar response to estrogens varies among different
species.

For example, in the mouse, estrogen-induced growth

has been reported to be limited to the duct system with no
lobule-alveolar development (Bradbury, 1932; Turner and Gomez,
193W •

In the rat estrogen induces a limited lobule-alveolar

development

(Turner and Schultz, 1931)> while in the guinea pig

estrogen will cause complete lobule-alveolar development

(Nel­

son, 1937).
With the development of synthetic estrogens a number of
workers have been able to produce fairly large milk yields in
dairy cows and goats from estrogenic treatment alone, which
suggests that this hormone may induce some lobale-alveolar
development.

(Mixner, Meites and Turner, 19l|l}., in goats;

Folley, Steward and Young, 19i|4* in cows; Walker and Stanley,
1940* in spayed heifers.)

5

Mixner and Turner (19l|3) reported that lobule-alveolar
development in the goat udder stimulated by diethyIstilbestrol
was not normal in appearance.

The alveoli were oversized and

papillomatous outgrowths of the epithelium projected into the
lumen.

However, when progesterone was administrated along with

diethylstilbestrol, normal alveolar development occurred.

It

should be pointed out that in these experiments and in those
reported by Nelson (1937) in the guinea pig, there is the pos­
sibility of intervention by extra-ovarian progesterone or other
steroids from the adrenal cortex.

Thus the ability of estrogens

to induce some lobule-alveolar growth does not rule out the need
for progesterone or progesterone-like compounds.
Progesterone
That the corpus luteum of pregnancy has a definite role
in developing the mammary gland was shown early by Nelson and
Pfiffner (1930, 1931)*

They were able to obtain lobule-

alveolar development in male and spayed female rabbits, rats
and guinea pigs with injections of crude corpus luteum ex­
tracts,

However, Turner and Schultze

(1931) observed that

crude progesterone itself had very little effect on mammary
growth unless estrogen was given first as a primer.
and Hill (193&) » on the other hand,

Gardner

showed that large aoses

of progesterone alone were capable of producing good lobulealveolar development in mice.

Mixner and Turner (19i|3) sug­

gested that the ability of small doses of progesterone to

j

6

elicit lobule-alveolar development in the presence of estrogen
was due to the action of estrogen in producing hyperaemia of
the mammary stroma, leading to an increased vascular perme­
ability.

This provides an easy access to the gland of proges­

terone and other mammary stimulating hormones needed for the
growing tissues.
Anterior Pituitary Hormones
The work of Corner (1930) and Lyons and Catchpole (1933)
demonstrated a mammary growth response to anterior pituitary
extracts, suggesting that this gland plays a role in normal
mammary development.

However, other workers failed to stimu­

late mammary growth with estrogen alone or with estrogenprogesterone combinations in hypophysectomized animals.
(Reece, Turner and Hill, 1937; Gomez and Turner, 1937a, in the
rat; Gomez and Turner, 1937a, in the guinea pig.)
As a result of the above findings Turner postulated the
’’mammogen theory . 11

He stated that the ovarian hormones, es­

trogen and progesterone, do not exert their growth stimulus
directly on the mammary gland but evoke their effects through
the anterior pituitary, which secretes a wduct-stimulating
hormone 11 and a f,lobule-alveolar hormone . 11

These were named

1fMammogen I,** the duct growth factor stimulated by the action
of estrogen, and "Mammogen II” the lobule-alveolar factor
elicited by progesterone stimulation.

To support this theory,

Turner and his students in a series of papers,

(Gomez and

7

Turner,

1937a; Gomez, Turner and Reece, 1937 ; Gomez and

Turner, 1938) showed that pituitaries from estrogen-primed
rats would induce mammary growth while pituitaries from un­
treated rats had no effect in the hypophysectomized male
guinea pig*

When pituitary extracts from pregnant cows were

injected into rats, a greater degree of mammary growth was
produces than with pituitary extracts from non-pregnant cows*
Nelson (191+1) and Folley et^ &1 (19i.p0) showed that topical ap­
plication of estrogens to rudimentary mammary glands of guinea
pigs and goats respectively, produced mammary development
while adjacent non-treated glands showed little or no growth*
This suggested that estrogens acted directly on the mammary
gland*

Mixner and Turner (191+3) interpreted these results to

mean that topical applications elicited a localized hyperaemia
in the gland which enabled sub-threshold levels of mammogens
to become effective*
Lyons

(191+2) reported convincing evidence that pituitary

prolactin may be a growth-stimulating factor by obtaining lo­
calized alveolar growth following intraductal injection of
prolactin in the teats of rabbits.

In hypophysectomized rats,

Lyons (191+3) was able to obtain well developed mammary glands
by injecting estrogen and prolactin combinations*

Lyons et al

(195?) showed that in hypophysectomized, gonadectomized imma­
ture male rats, it was possible to produce different degrees
of development of the female mammary glands by the following
sequence of injections:

(1 ) estrone and growth hormone

8

produced mammary duct growth,

(2 ) estrone,

progesterone and

prolactin produced lobule-alveolar growth comparable to early
pregnancy,

(3 ) estrone, progesterone, lactogenic and growth

hormone produced lobule-alveolar development comparable to
late pregnancy,

(I4.) prolactin, growth hormone and hydrocortisone

acetate initiated milk secretion in the fully developed glands.
It seems logical to conclude therefore, that the ovarian hor­
mones stimulate mammary growth best in the presence of anterior
pituitary hormones.

The "mammogenic hormones 11 of the anterior

pituitary appear to be prolactin, ACTH and growth hormone.
Adrenal Cortex
The role of the adrenal cortex in mammary development
is not yet fully understood.

Trentin and Turner (19JLp7) reported

that adrenalectomy reduced mammary growth in rats, while Smithcors and Johnston (l9 lj-8 ) found an increase in mammary growth
in adrenalectomized rats.

Van Heuverswyn ot_ al (1939) and

Nelson (1937) noted that desoxycorticosterone

(DCA) stimulated

duct growth in mice and guinea pigs respectively.

Mixner and

Turner (191p3) reported that DCA had about l/3 the mammary growth
activity of progesterone In mice.

Turner and Meites

(19lp7)

observed that DCA did not augment the pituitary prolactin con­
tent in female rats.
Recently the mammary growth effects of ACTH and the
glucocorticoids have become the subject of investigation.
Nelson (l9ipl) noted that crude ACTH preparations stimulated

9

mammary growth in hypophysectomized rats.

However, Flux (1954)

observed that cortisone, hydrocortisone and ACTH produced in­
hibition or mammary growth when injected into estrone-treated, ^
ovariectomized mice of the CHI strain.

When given alone, ACTH

and the glucocorticoids were without effect on the mammary
glands of ovariectomized mice.
In contrast Selye

(1954®-) found substantial mammary

development and secretory activity when hydrocortisone and ACTH
were injected into ovariectomized rats,
estradiol.

Selye

previously primed with

(1954^) further reported that adrenalectomized

and ovariectomized rats treated with hydrocortisone and estra­
diol showed marked mammary development and secretion.

When

hydrocortisone was given alone, mammary growth was reduced and
confined to the ducts.
Hohn (1957) has recently reported that In the adrenalectomized guinea pig, estrone alone was incapable of producing
lobule-alveolar development and elicited only duct growth,

sug­

gesting an adrenal involvement in mammary growth by estrogen.
The hormones responsible for the lobule-alveolar growth of the
mammary gland may be progesterone or other adrenal-cortical
hormones with progesterone-like activity.

This probably e x ­

plains why Nelson (1937) was able to obtain full mammary growth
in the intact guinea pig with estrogen Injections alone.

Involution of the M ammary Gland during Lactation
Failure to remove milk from the mammary glands leads
to rapid disintegration of the lactating glad to a resting
condition, which closely resembles that seen in virgin ani­
mals *

Williams (l9lj-2) noted that In the mouse there is a

rapid engorgement of the gland with secretion upon the removal
of the young*

This is followed by absorption of the milk.

By

the third day after removal of the young the mammary glands of
the

dams

are still morphologically intact*

However, from the

fourth day onward there is a rapid regression of the paren­
chyma*

The alveoli collapse and the small interlobular ducts

disintegrate, leaving the collapsed alveoli as isolated cell
masses*

Finally the site of the alveoli and small ducts are

infiltrated with corpuscles.

The larger ducts show very little

degeneration but are reduced in size*
Cessation of milking or the removal of young are not
the only factors responsible for mammary involution.
the peak of lactation has been reached,

After

a period of decline

follows in which secretory activity gradually decreases
despite the fact that the animal still produces milk.

Whether

this is due to decrease in hormones stimulating lactation,
decreased secretory rate of the individual alveoli, or to
degeneration of the alveoli is not definitely known.

Selye and McKeown (193^) observed that despite a
strong and continuous suckling stimulus, the mammary glands of

11

mice will involute over a period of time.

Turner and Reineke

(1 9 3 6 ) reported that in advanced stages of lactation in the
goat, involution was almost complete with only a small number
of active alveoli still present.

When milking was suspended

on one side of the udder of an actively lactating goat, while
continuing milking on the other side, they observed that in­
volution was retarded in the side not milked.

This seems to

demonstrate that the milking stimulus is necessary to maintain
the integrity of the gland,
Selye (193U) also demonstrated that suckling is of prime
importance in preventing the rapid involution of the mammary
gland.

If he ligated the galactophores of a rat, thus pre­

venting milk from being withdrawn, and permitted active suckling
by the young, rapid involution did not take place.

When some

of the nipples were excised to prevent suckling, mammary invo­
lution was retarded provided the other nipples were suckled.
Hooker and Williams (19JL}.0, 19i|l) observed the same phenomenon
in mice although they did note some involution that was not
characteristic of post-weaning involution.
Hooker and Williams (l9ipl) applied turpentine to the
nipples of lactating mice isolated from their young in an at­
tempt to mimic the suckling stimulus by irritation.

They re­

ported that mammary involution was retarded in the treated
glands and even in the untreated glands to a lesser degree.
Reece and Turner (1937a) reported that pituitary pro­
lactin content decreased in cows upon resumption of milking

12

after a period of milking*

This was also demonstrated to be

true when the galactophores in rats were ligated, thus showing
that decreased milk secretion was related to the milking stimu­
lus rather than to the removal of milk from the gland*

It was

further demonstrated that suckling in rats and rabbits main­
tained a higher prolactin secretion than in unsuckled animals
(Meites and Turner, 19i|-8b) *
Williams and Hooker (19i.pl) and Williams (191+5) showed
that prolactin injections of 20-60 I# U. daily retarded mam­
mary involution In mice after the young were removed*

Pro­

lactin maintained lobule-alveolar and duct system similarly
to mammary gland of mice at parturition when Injected over a
seven-day period.
That the suckling stimulus influences the release of
other anterior pituitary hormones than prolactin has been
demonstrated by Desclin (19^4-7) •

He showed that when rats

were spayed at parturition and the galactophores were
ligated, the appearance of castration cells in the pituitary
could be prevented if suckling was continued*

Gregoire

(19^7)

reported that thymic involution resulting from gestation could
be maintained in rats spayed at parturition if the suckling
stimulus was continued*

13

Factors Controlling the Initiation and
Maintenance of Lactation
Role of the Anterior Pituitary
Strieker and Grueter (1928) were the first to demonstrate
that lactation could be initiated in ovariectomized, pseudo­
pregnant rabbits by injections of anterior pituitary extracts*
This was the first step in demonstrating that lactation was
not only inhibited by the products of pregnancy, but a definite
hormonal stimulus was required for the initiation of lactation*
The factor necessary for this stimulus was demonstrated to be
prolactin (Riddle and Bates, 1939; Lyons,
191-4-8).

1914-2

; Meites and Turner,*

The latter two groups of workers showed that small quan­

tities of prolactin injected into the galactophores of nonlactating rabbits with fully developed mammary glands evoked a
localized lactation, indicating that prolactin acts directly
on the epithelial cells*
The reports of several workers

(Gomez and Turner, 1938,

1937b; Nelson and Gaunt, 1938, 1937b) suggested that other
anterior pituitary hormones than prolactin were necessary for
the initiation of lactation in hypophysectomized animals since
crude extracts of the anterior pituitary initiated lactation
in such animals while purified prolactin extracts did not.
ACTH appears to be one of these hormones, since lactation can
be initiated In hypophysectomized guinea pigs with purified
prolactin extracts and ACTH or adrenal cortical extracts (Gomez
and Turner, 1938, 1937b; Nelson and Gaunt, 1937b)*

Recently

Lyons et al (1953) have shown in hypophysectomized rats that
lactation can be initiated with combinations of prolactin,
growth hormone and cortisone or ACTH, provided the mammary
glands are fully developed.

This is further proof of the

necessity for other pituitary hormones than prolactin to ini­
tiate lactation in hypophysectomized animals.
however,

Prolactin alone

can initiate lactation in intact animals with develop

mammary glands, with the possible exception of the rat (Meites
and Turner, 19l|-8)*
Adrenal Cortex
It has been clearly established that adrenalectomy
interrupts established lactation.

Adrenalectomy before par­

turition does not prevent the Initiation of lactation but
lactation will not be maintained (Gaunt, 1933; Carr, 1931)*
However, there is some disagreement as to which cortical
steroids are responsible for the maintenance of lactation.
Gaunt et al (19i|2) reported that cortisone was able to com­
pletely maintain lactation in rats adrenalectomized the day
after parturition.

However, DCA only partially maintained

lactation.
Folley and Cowie

(I9l|i;) and Cowie and Folley (l9l|-7)

have shown that DCA is more effective than cortisone in main­
taining lactation in rats adrenalectomized on the fourth day
after parturition.

However, there was not a complete restora­

tion of lactation with these compounds when given individually

15

Even when dietary protein was increased by 50 percent, so as
to favor the action of the glucocorticoids, the lactational
response was subnormal*

Cowie

(1952) was able to obtain com­

plete maintenance of lactation in adrenalectomized rats by
the implantation of cortisone and DCA pellets*

Reece (1939)

initiated a higher degree of milk secretion (++♦) in pseudo-

^

pregnant rats when prolactin was given together with adrenal
cortical hormones and

20

percent glucose solution, than with

prolactin alone (++).
Brownell, Lockwood and Hartman (1933) postulated the
existence of a specific adrenal cortical hormone, !,cortilactin*”
However, Hurst, Meites and Turner (19I4.2 ) were unable to detect
any pigeon crop-stimulating activity in adrenal extracts from
several species.
The circulating levels of the adrenal corticoids (17hydroxicorticosteroids) begin to rise early in pregnancy and
remain high until shortly after parturition (Gemzell, 1953)*
Venning (19l|-6) has also shown a progressive rise in excretion
of the glucocorticoids in women beginning early in pregnancy.
There is a significant reduction of circulating
leukocytes during suckling in the lactating rat and mouse.
However, this may be due to removal of leukocytes via the
milk.

Tabachnick and Trentin (1951) have suggested a possible

involvement of the adrenal in this lymphopenia of lactation,
since they found a significant decrease in the mean ascorbic
acid of the adrenals in suckled as compared to non-suckled mice.

16

Folley and his group reported the effects of hormones
on the metabolism of mammary gland slices in vitro.

After

parturition the R. Q. of the mammary gland in the presence of
glucose or glucose plus acetate was above unity (Folley and
French, 1914-9).

A composite respiratory curve showed that mam­

mary gland slices from

20

-day pregnant rats, incubated in bi­

carbonate saline buffer with glucose plus acetate added,
produced an over-all fall in pressure while mammary tissue from
lactating rats exhibited a rise in this curve (Balmain and
Folley, 1952).

When prolactin was added to mammary gland

slices from rats pregnant

20

days there was no effect on

respiration.

However, cortisone produced an increase in

respiration.

Cortisone and prolactin did not give any greater

increase in respiration than cortisone alone.
These English investigators further found that prolac­
tin added to mammary slices of rats

1-5

days after parturition

elicited an increase in respiratory activity.

They suggested

that cortisone conditions the mammary gland before parturition
and that after parturition the gland is able to respond to
prolactin.

This remains to be proved.

Suckling Reflex
Suckling is known to evoke the release of oxytocin
through a neurohormonal reflex (Ely and Peterson,

19^1; Cross,

1955&* h). Reece and Turner (1937) have also demonstrated that
suckling results in release of prolactin from the pituitary,

17

and Meites and Turner (191^-8) showed that suckled rats and rab­
bits contained more prolactin in their pituitaries than non­
suckled animals*
of lactation.

Suckling is not necessary for the initiation

At parturition, although neither maximum milk

production nor maximum pituitary prolactin content is attained
without it.
Benson and Folley (1958) reported that release of pro­
lactin could be stimulated by treatment with oxytocin, as
judged by the appearance of secretion in the mammary gland.
However, they did not actually measure prolactin secretion.
These investigators inhibited the decline in milk secretion in
rats which had their young removed, by injecting I I . U. of
oxytocin three times daily from the fourth to the thirteenth
day after parturition.

They suggested that impulses arising

from sensory fibers of the mammary gland stimulates the poster­
ior pituitary to release oxytocin, which in turn produces a
release of prolactin.
Attempts to Increase Lactation (Galactopciesis) or
Maintain Established Lactation with Hormones
It has been established that lactation in most mammals
rapidly reaches a peak and then slowly declines over a long
period of time.

Attempts have been made to Increase milk yield

during lactation by the use of hormones.

Folley and Young

(1939) have classified hormones with this ability as "galactopoietic.,t

It is important to emphasize that these "galactopcietic”

18

agents may not necessarily play a role in the initiation of
lactation (lactogenesis)•
Azimov and Krouze

(1937) were the first to demonstrate

galactopoietic activity in crude anterior pituitary extracts
during the declining phase of lactation in cattle.

They showed

that injections of ox anterior pituitary into cows produced a
marked but temporary increase in milk (lasting

5

to

6

days).

Folley and Young (193$) confirmed this work in cows, using
purified prolactin and growth hormone preparations.

These

workers found that a single injection produced a mean increase
in milk yield of about

percent.

10

Folley and Young (1939, 191+0) were able to substantially
increase yields in cows by

15-20

percent by injecting crude ox

pituitary extracts every other day over a three-week period.
The lactation curves obtained by pituitary treatment during
the declining phase of lactation were identical in slope with
untreated cows.

They also showed that crude pituitary extracts

did not affect the peak of lactation nor delay the onset of the
decline of lactation.
Cotes et al {191+9) showed that growth hormone given as
a single injection of
milk yields.

30

mg in cows, substantially increased

This was confirmed by Donker and Petersen (1951)*

Shaw (1955) treated a few cows at parturition with growth hor­
mone and was able to increase lactation during the entire lac­
tational curve over that of untreated control cows.
ACTH injections exerted an inhibitory effect on lacta­
tion in cows (Cotes e_t al, 191+9)*

This action does not seem to

19

be in agreement with the view that ACTH and the adrenal-cortical
hormones are necessary Tor the maintenance of lactation in the
adrenalectomized animals described earlier*
ages used may have influenced these results.

However, the dos­
Roy (I9i|7) claimed

that large doses of purified ACTH injected every other day, in­
creased lactation during the declining phase as much as
percent.

20

When prolactin was added to the ACTH preparation there

was no significant increase above that of ACTH alone.

Hater

wcrk by Folley (1955) indicated that ACTH is only capable of
inhibiting rather than augmenting lactation in cows.

20

EXPERIMENT I.

EFFECTS OF ACTH AND ADRENAL CORTICAL
HORMONES ON MAMMARY GROWTH AND PITUITARY
PROLACTIN CONTENT IN INTACT RATS
Procedure

This experiment was performed to determine the effects
of cortisone, hydrocortisone and ACTH on mammary development
and pituitary prolactin content in intact female rats.
Forty-two mature female albino rats, of the Carworth
strain, were divided into four groups and injected daily for
10

days as follows:

1

, controls,

0-85

percent saline;

2

,

2

mg

cortisone; 3> 2 mg hydrocortisone; and If, 2 mg (2 I- U.) ACTH
(Armourfs).
volumes.

All injections were made subcutaneously in 0.1 cc

On the 11th day the rats were killed and the pitui-

taries were removed, weighed and prepared for prolactin assayThe prolactin content of the pituitaries from each
group of rats was assayed in 5 or

6

white Carneau pigeons by

the sensitive intradermal method of Reece and Turner (1937)In one group of

6

pigeons, the control pituitaries were injected

over one crop sac and directly compared with the pituitaries
from the hydrocortisone-treated rats, injected over the other
crop sec.

In another group of 5 pigeons, the pituitary sus­

pension from the cortisone-treated rats was directly compared
with the pituitaries from the ACTH-treated rats.
of

2

Thus a total

rat pituitaries were injected over each crop sac during a

21

four-day period.

On the

5

th day the pigeons were killed and

the crop glands were removed and rated visually for degree of
proliferation.
The right inguinal mammary gland was dissected from
each rat and prepared for gross mounting and for fixing in
Bouinfs Fluid.

Standard histological staining procedures were

employed, using hematoloxylin and eosin.

Each of the excised

mammary glands was rated (0 —2_p) for degree of development and
secretion.
Results
These findings are summarized in Table 1 and Figure

1

.

Cortisone and particularly hydrocortisone inhibited body growth
while the dose of ACTH employed had no effect on body growth.
The mammary glands of the controls showed mostly bare ducts
with little branching, and few to moderate number of ductal
buds.

Cortisone and hydrocortisone induced marked branching

of the ducts and considerable lobule-alveolar development.
| ACTH was least

effective in eliciting

mammary growth, but pro­

duced moderate

branching of the ducts

and limited lobule-

alveolar growth.
Cortisone was the most effective in inducing secretory
activity in the mammary glands while hydrocortisone was only
moderately effective and ACTH was least effective.
no evidence of

secretion in the control glands.

mammary glands

are shown in Figure 1.

There was

Representative

22

The pituitary weights were not altered by any of the
hormone treatments when compared on a body weight basis*
However, all three hormones increased the prolactin content
of the pituitary.

Cortisone and hydrocortisone increased it

about 23 and Ipl percent respectively, and ACTH about 71 percent
(on a body weight basis).

23

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24

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25

EXPERIMENT II.

EFFECTS OF ESTRONE AND CORTISONE ON
PITUITARY PROLACTIN CONTENT AND MAMMARY
GROWTH OF 0VARIECTOMIZED RATS
Procedure

This experiment was performed In an attempt to compare
the results of the previous experiment in intact rats with
ovariectomized rats, and also to see whether there was any
interaction between estrogen and cortisone on pituitary pro­
lactin content and mammary growth.

One hundred mature female

albino rats of the Carworth strain were ovariectomized.

After

a period of Ilf. days the rats were divided by weight into eight
uniform groups of 10 rats each.

These were further divided

into two major groups of lf.0 rats each (four groups of 10), and
the remaining two groups (20 rats) were used for controls.

The

treatments of the groups were as follows:
(1) controls, saline injections only;
1 mg. cortisone acetate only.

(2) controls,

The two major groups received

(1 ) 5 u g • of estrone, and (2) 10 ug. of estrone.
two major groups,

cortisone was administered to the four sub­

groups as follows:
cortisone daily;
cortisone daily.

Within these

(l) no cortisone, saline only;

(3 ) 2 mg. cortisone daily; and (i|)

(2) 1 mg.
mg.

All injections were given subcutaneously in

0.1 cc. volumes of physiological saline, daily, for a period
of 10 days.

On the 11th day the rats were killed and the

pituitaries were removed and prepared for prolactin assay.

26

The prolactin content of the pituitaries was assayed
in 25 white Carneau pigeons by the sensitive intradermal method
of Reece and Turner (1937)*

These were divided into 5 groups

of 5 pigeons each and were Injected as follows*

(l) control

pituitaries were directly compared with pituitaries from
estrone-treated

(5

ug.) rats;

(2 ) pituitaries from cortisone-

treated rats were directly compared with the pituitaries from
estrone

(5

ug.) and cortisone-treated (l mg) rats;

taries from estrone

(5

ug*) and cortisone-treated

(3 ) pitui­
(2

mg.) rats

were directly compared with pituitaries from the estrone
and cortisone-treated (I4. mg.) rats;
treated

(10

(l|.) pituitaries from estrone-

ug.) and cortisone-treated

and (5) pituitaries from estrone
(2

ug*)

u g . ) rats were directly compared with pituitaries

(10

from the estrone

treated

(5

(10

(1

mg.) rats;

ug.) and cortisone-

mg.) rats were directly compared with pituitaries

from the estrone

(10

ug.) and cortisone-treated (1^. mg.) rats.

A total of 2 rat pituitaries were Injected over each
crop sac for a period of four days.

On the 5th day the pigeons

were killed and the crop glands were rated for degree of pro­
liferation.

The right inguinal mammary gland from each rat

was removed, stretched gently on a small piece of cork, fixed
in Bouin* s fluid over night and prepared for histological
examination.

27

Results
The mammary glands of the controls (group 1) showed
duct growth and budding, while the cortisone-treated (l mg.)
controls (group 2) showed a larger number of ductal buds and
some degree of lobule-alveolar development.

The rats receiving

estrone alone (groups 3 and 7) exhibited duct growth only.

How­

ever when cortisone was administered to the estrogen-treated
rats (groups Ip, 5» 6, 8, 9, and 10), lobule-alveolar development
j
was substantially stimulated by all levels of cortisone.

A con­

siderable amount of secretion was noted in the mammary glands
of the rats treated with

cortisone

and estrone.

mammary glands are shown

in Figure

3*

It can be seen in
gain in the control rats

Representative

Table 2 that there was a good weight
(group 1)and

in those animals which

received the lower levels of cortisone (groups ip and 8).

How­

ever, at the higher levels of cortisone (groups 5 and 9), there
was a definite inhibition of body growth, and at the highest
level of cortisone

(groups 6 and 10) an actual loss of body

weight•
When pituitary prolactin contents were compared, no
significant change was found between the controls given saline
only (group 1) or those injected with 1 mg. of cortisone (group
2).

Estrone alone (groups 3 and 7) produced a definite increase

In pituitary prolactin content at both levels (5 ug. and 10 ug.)
over that of the controls (groups 1 and 2).

When 1 mg. of cor­

tisone was added to the estrone (groupsk find 8) the pituitary

28

prolactin content was significantly augmented above that pro­
duced by estrone alone (groups 3 ar*d 7)«

Only the largest

dose of cortisone employed (group 10) inhibited the action of
estrone in increasing pituitary prolactin content.

29

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Pacing page 30

Figure

2.

Effect of estrone and cortisone on
pituitary prolactin content.

1

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Pacing page 31

Figure 3»

Histol ogi cal sections of representative
mammary glands (QOx)*
(1)
(2)
(3)
(U

0.8$ percent saline
10 u g * estrone
1 mg, cortisone
io ug. estrone and 1 mg,

cortisone

31

Figure 3®

32

EXPERIMENT III.

EFFECTS OF CORTISONE ON GALACTOPOIESIS
IN THE RAT
Procedure

From the results of the previous experiments it seemed
reasonable to assume that if cortisone had the ability to in­
crease pituitary prolactin content, it might also produce a
galactopcietic effect in the suckling rat. ( It is extremely
difficult to accurately measure the lactational response in
small laboratory animals when compared to larger animals such
as the cow and goat.

Since it is difficult to actually measure

milk output, it is necessary to employ survival of litters and
litter growth curves as measures.
ter were employed.

In this experiment the lat­

However, it should be pointed out that

this Is not an absolute but only a relative index of lactational
response.

In litter growth curves the daily milk yield is not

directly calculable, since the daily weight loss due to excreta
and insensible perspiration are not measured.
Cowie (191+6) used a logistic equation and plotted a
mean growth curve for litters of lactating dams from birth to
16 days of age and obtained a sigmoid curve.
^

However from the

6th to the 11th days of lactation, the points on this curve
were in a straight line, and he therefore claimed that during
this 5-day period the mean total increase in litter weight was
approximately constant as well as at a maximum for each rat.

j

33

This gain was termed "litter growth index" and he used it as a
quantitative measure for lactational responses in rats.

For

reasons pointed out above, this is not believed to be a true
reflection of milk yield but is estimated by Cowie to be roughly
50-60 percent of the true output.
Thus, if this period (6th to 10th day) of lactation
represents the peak of lactation in rats, the two extremes of
this sigmoid curve could then be called the initial phase of
the declining curve of lactation.

The average litter weight

gain in the present experiment was divided into three periods
for comparison as follows:
(2)

(l) initial phase (0-5th day);

peak phase, Cowie1s "litter growth index" (6th-10th day);

and (3 ) declining phase (llth-l8th day).
Brody (191*5) took into consideration the growth rate
of an animal at a given age and related it to body size at a
given age, and developed a logarithmic equation for showing
growth rate of an animal during a given unit of time.
v

*

In W« - In W-,
c.
X
t2 - t

k

*

In

or simplified,
Wo

* t

wi
where

k

w2

“ dally instantaneous rate of growth
a= ratio of the surviving proportion of litter weights

Wi
t

* time interval in days between observations.

3k

The "instantaneous growth rate" index has been used in
the present experiment merely as a refinement of previous
measurements and to see to what extent it agrees with them.
Thirty mature female albino rats of the Carworth strain
were bred.

Once pregnancy was established, the dams were placed

in individual cages.

These cages were designed to make the

dam's ration inaccessible to the young throughout the experi­
mental post-partum period of 18 days.

This insured that the

young received food only from their mothers.

On the day of

parturition the litters were reduced to 7 young each and these
were randomly grouped as follows:
only;

(2)

0.25

(1) controls (9 rats), saline

mg. cortisone (8 rats);

(7 rats); and (Ip) 1.0 mg. cortisone

(3) 0.5 mg. cortisone

(6 rats).

All injections

were administered subcutaneously, daily, for the eighteen-day
period after parturition.

The litters were weighed daily and

the dams weekly until termination of the experiment.

On the

19th day the dams were killed and the adrenals were removed
and weighed on a Roller-Smith balance.
Results
It can be seen in Table 3 that there was no significant
weight loss in the control dams (group 1) during the experi­
mental period.

However, in each of the three treated groups

(groups 2, 3 and i|.) there was a significant weight loss of
approximately

18

, 19 and lp6 grams each, respectively.

35

In the rats receiving 0*5 mg. cortisone

(group 3)

the average litter weight gain during the experimental period
was significantly increased over that of the controls (group l).
Groups 2 and ip, which receiver 0.25 mg. cortisone and 1.0 rag.
cortisone each daily, exhibited no significant litter weight
gain over the controls.

However, it is interesting to note

that even though the dams receiving 1.0 rag. cortisone showed
a larger body-weight loss, they still maintained their young
at the same or at a slightly higher growth rate than in the
control rats.
Table ip shows a somewhat more complete picture of the
lactational response in these rats.

The rats receiving 0.5

mg. cortisone (group 3) showed no effect during the initial
phase of lactation (0-5th day).

However, significant increases

over that of the controls can be seen in this group during the
peak of lactation (6th-10th days and during the declining phase
(llth-icth days) •
tisone

Although the rats which received 1.0 mg. cor­

(group Ip) did not show a significant litter weight gain

over the controls during the entire l8-day experimental period,
they did exhibit a significant Increase during the declining
phase of lactation (llth-lGth day) .

When "instantaneous growth

rates” are employed this same trend is noted (Table 5)*

Figure

Ip is a graphical representation of these experimental data.
These results suggest that cortisone may increase the lactational
response in the intact female lactating rat.

36

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Facing page 39

Figure If..

Effects of cortisone on litter weight
gains in the rat*

39

Total
( 1 8 days)

f
I

"[

Initial
(o-5 d.,.)

mmsm
m

1

Peak
(6 - 1 0
days)
“Litter Growth Index 11
^
____ ___
sard.,.)
^

2 lj.O

160

120

Average

Body

Weight

in

Grains

200

m

Control

Cortisone Cortisone
0*25 mg.
0.5 mg.

Figure Ij.

Cortisone
1 . 0 mg.

ko

EXPERIMENT IV.

EFFECTS OF CORTISONE, GROWTH HORMONE,
PROLACTIN, OXYTOCIN AND ACTH ON LACTATION
AND INVOLUTION OF MAMMARY GLANDS IN RATS
Procedure

This experiment was performed to determine whether
cortisone as well as growth hormone, prolactin, oxytocin and
ACTH could exert galactopoietlc effects and retard mammary
involution in lactating rats.
In this experiment 9i+. mature female rats of the Carworth
strain were used.

The experimental design was the same as in

Experiment III except for the following modificationsi
parturition the litters were reduced to
of

7

6

(l) at

young each instead

, in order to enable a larger percentage of litter sur­

vival and to insure that at least this many were alive on the
day of parturition.

(2 ) All injections were made subcutan-

eously once daily, with the exception of oxytocin which was
injected twice daily.
additional

10

The injections were carried out for an

days (l8 th- 2 8 th day) after removal of the young.

(3) On the 28th day, the dams were killed and the right in­
guinal mammary glands were removed and prepared for histologi­
cal examination.

(Ip) The control group was divided into two

sub-groups, one (group l) of which was used to determine mammary
involution at the end of the
the other (group

2

28

-day experimental period while

) was used to follow normal daily involutionary

41

changes of the mammary glands from the

1 8 th

to the 27th day

(see Appendix, Figures 14-23)*
The total number of litters reported in the data are
68

and these were treated as follows:

only (17 rats);
0.5

mg*

(8

lactin,

1

(2 ) cortisone,

rats);
mg.

(8

rats)

tion);

rag.

(8

(4 ) growth hormone,
rats);

1

rats);

(3) cortisone,

mg*

rats);

(8

rats);

(5 ) pro­

mg. cortisone,

1

(7) oxytocin, 1 I. U.

(this was injected twice daily,

(8 ) ACTH, 1 mg.

(8

(6 ) growth hormone,

0.5 mg. and prolactin, 1 mg.
(6

1

(1 ) controls, saline

0*5

1

. U. per injec­

(5 rats).

An additional group of

8

rats were added to the latter

control series in order to further determine the degree of
daily involution after removal of the young on the l8 th day.
Only litters which were healthy at
a measure of milk secretion.

18

days of age were used as

Dams which developed infection

were discarded.
The control groups (1 and 2) and the two groups given
cortisone

(3

and

4

)

not develop any infection in the dams

and the young were all normal at 18 days.

The dams in some

of the other groups developed abnormal litters as follows:
growth hormone
(group

6

(group

5

)# six litters were stunted; prolactin,

), two litters were stunted, and one dam was killed

because of infection; growth hormone, cortisone and prolactin
(group

7

), three stunted litters; oxytocin (group

8

), three

litters were stunted and ACTH (group 9)> two litters were
stunted and one dam died of infection.

k2

Results
As can be seen in Table

6

, there was no significant

weight loss in the control dams (groups
entire experimental period
sone (groups

3

days).

(28

1

and

2

) over the

Both levels of corti­

and ip) produced a weight loss.

However, this

was not as great as that exhibited in Experiment III.

Both

growth hormone (group 5) and ACTH (group 9) increased the body
weight of the dams.

The other groups showed no significant

changes in body weight.
In the rats

receiving cortisone, prolactin, oxytocin or

ACTH (groups 3* 4*

6

weight gain during

the experimental period

0.5

#

8

and 9), there was a significant litter
(18

days), while

mg. cortisone (group ip) produced the greatest increase in

litter weight gain over that of the controls.
ceiving growth hormone (group
in litter weight.
mones (group

7

5

In the rats re­

) there was no significant gain

The rats receiving the combination of hor­

) also showed a significant increase in the average

litter weight gain, but it was no greater than that in the rats
given 0.5 mg. cortisone (group ip).

Therefore, prolactin and

cortisone do not seem to have a synergistic action on milk
secretion as judged by gain in weight of the young.
The results in Table 7 show that cortisone at the 0.5
mg. level (group ip) only slightly increased lactation during
the initial phase (0-5th day).

However, milk secretion was

significantly increased between the
the

7

6

th and

10

th day and during

declining phase (llth-l 8 th day) over that of the controls

(groups 1 and 2).

The rats treated

with all three hormones

(group 7) and the prolactin-treated

rats (group

the same trends as the rats treated

with O.j?cortisone

(group I4.) •

Growth hormone

(group 5 )» on

6

) showed

the other hand, re­

sulted in no greater gains in litter weight during these
periods than in the controls (groups

1

and

2

).

It is interesting to note the results obtained with
oxytocin (group

8

).and ACTH (group 9)«

Although their stimu­

lating effect on lactation was similar to that of cortisone,
prolactin or the hormone-combination group during the peak
phase and declining phase of lactation, there was no effect
at all during the initial phase of lactation.
When computed in terms of "instantaneous growth rates,"
the same trends were noted in litter weight gain (Table

8

)•

Figure £ shows a graphical representation of these experimental
data.
Histological examination of the mammary glands reveal
striking differences between the different groups (Figures
6-13).

The control mammary glands (Figure

involution.

6

) show marked

All the alveoli are collapsed or in an advanced

state of disintegration.

Also, marked fibrosis of the lobules

can be seen, despite an apparently normal ductal epithelium.
Cortisone

(Figures 7 and

8

), prolactin (Figure 10), and the

hormone-combination group (Figure 11) showed definite mainten­
ance of alveolar structure as compared to the controls.

In the above hormone-treated groups the alveolar epi­
thelium was intact in most of1 the sections cut# although some
degeneration was noted in a few lobules.

The greatest amount

of degeneration can be seen in the cortisone-treated (0 . 5 mg.)
rats (Figure

8

)•

these sections.

Inspissated secretions were seen throughout
These mammary glands

(groups 3, ip,

6

and 7)

were comparable to control mammary glands from rats five days
after the young had been removed.

Cortisone at the 0.5 mg.

level (group Ip) produced mammary glands which resembled a
control mammary gland six days after the young we're removed
(see Appendix, Figures 19 and 20).
Growth hormone, oxytocin and ACTH appeared to have,little
or no effect in retarding involution of the mammary gl^nd.
Sections from the mammary glands of these rats showed no marked
differences from those in the controls (Figures 9,

12

-

and 13).

The oxytocin and ACTH-treated groups did show some very sparse
but intact alveoli.

However,

this was also seen in some con­

trol glands.
Macroscopic examination of the mammary glands from all
groups revealed very few differences at the end of the
post-weaning period.
hormones,

1 0 -day

In the controls and all rats treated w i t h W

the larger lobular ducts were intact.

However, the

smaller interlobular ducts had disintegrated and the lobules
were arranged in clusters throughout the parenchyma.
cortisone-treated rats (groups ip and
(group

6

5

)* in

In the

prolactin rats

), and in the rats given the combination of hormones

45

(group 7 ) f the lobules appeared more dense and the individual
alveoli were larger than those in the control group.
These results show a similar galactopoiettc activity for
cortisone as in ExperimentfIII, but in addition indicate that
prolactin, ACTH and oxytocin also possess an ability to in­
crease lactation.

They also demonstrate that cortisone and

prolactin were able to markedly retard mammary involution.

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Facing page ip9

Figure 5

Effects of cortisone, growth, hormone,
prolactin, oxytocin and A C T H on body
weights of litters.

1+9

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Figure

6

.

^0

Control mammary gland f r o m a rat 10 days
after young have been removed.
Top 1 3 0 x,
bottom 250x.
Stained with hematoxylin
and eosin.

50

Figure 6

Facing page 51

Figure 7.

Mammary gland from a rat receiving
1 . 0 mg cortisone daily for 1 0 days
after young were removed#
Top 1 3 0 x,
bottom 250x.
Stained w ith he matoxylin
and eosin.

51

w

m

m

Figure ?•

Facing page 52

Figure

8

.

Mammary gland from a rat receiving
0 . 5 mg. cortisone for 1 0 days after
young were removed.
Top 130x,
bottom 250x.
Stained with, hematoxylin
and eosin.

52

Figure 8*

Facing page 53

Figure

9.

Mammary gland of a rat receiving
1 mg* growth hormone for 10 days
after young were removed*
Top 1 3 0 x,
bottom 250x.
Stained witn h e matoxylin
ana eosin •

53

st

Figure 9

Facing page

Figure 10♦

SU

Mammary gland of a rat receiving
1 mg. prolactin aaily for 10 ciays
after young were removed.
Top 130^
bottom 250x.
Stained with hematoxy
and eosin.

54

Figure 10

F a c in g

Figure

11.

page

55

Mammary glanu of a rat receiving
cortisone 0.5
prolac tin 1.0
mg . and growth, hormone 1.0 mg.
Tor 10 days after young were r e ­
moved.
Top 130x, bottom 250x.
Stained with hematoxylin and eosin.

55

Figure 11•

F a c in g

Figure

12.

page

$6

Mammary gland of a rat receiving
oxytocin 0.5 1* ^ » twice aaily,
Tor ten aajs after young were r e ­
moved.
Top 130x, bottom 250x.
Stained with hem atoxylin and eosin.

56

Figure 12*

F a c in g

Figure

13*

page

57

Mammary gland of a rat receiving A C T H
1 mg* for 10 days after young were r e ­
moved.
Top 130x, bottom 250x.
Stained
with hematoxylin and eosin.

57

Figure 13»

58

EXPERIMENT V.

FACTORS INFLUENCING THE RELEASE OF
PROLACTIN IN SUCKLING MOTHER RATS
Procedure

The object of this experiment was to determine whether
injections of pitocin or a nervous stimulation which produced
pitocin release from the pituitary (electrical stimulation of
the cervix) results
lactating rats.

in release of pituitary prolactin of

For this investigation, 25 mature female

albino rats of the Carworth strain were bred.
day after parturition,
and were divided into
as follows:

On the third

the dams were removed from their young
groups of 5 each.

(l) Negative controls.

These were treated

The dams were separated

from their young for 15 hours and were then killed.
tive controls.

(2) Posi­

After 12 hours of separation from their young,

the mothers were returned to their litters for a period of
three hours of suckling, at the end of which time the dams
were killed.

(3) After 12 hours of separation from their young,

the cervices of the mother rats were electrically stimulated
for a period of one minute with 25 volts, on the 12th,
lipth and 15th hours.
the last stimulation.
their young,

1 3 th,

The dams were killed immediately after
(ip) After 12 hours of separation from

these rats were Injected subcutaneously with 1.0

IT, pitocin on the 12th, 13th, llpth and 15th hours.
dams were killed immediately after the last injection.

The

59

In all four* groups} the pituitaries were removed from
the mother rats immediately after sacrifice on the
and were weighed and prepared for prolactin assay.

15

th hour,

The prolac­

tin content of the pituitaries from each group of rats was
assayed in 20 white Carneau pigeons by the intradermal method
of Reece and Turner (1937).
In one group of 10 pigeons, the 'positive control (group
2) pituitaries were injected over one crop sac and directly
compared with the pituitaries from the electrically stimulated
rats (group 3), injected over the other crop sac.

In another

group of 10 pigeons, the pituitary suspension from the negative
control rats (group i) were directly compared with the pitui­
taries from the pitocin-treated rats (group i|).

One-quarter

of a pituitary was injected over each crop sac during a Jq-day
period.

On the 5th day the pigeons were killed and the crop

glands were removed and rated visually for degree of prolifer­
ation.
Results
The pituitary weights, when compared on a body weight
basis, did not show any significant alteration as a result of
the different treatments.

The negative controls (group l)

showed a significant increase in prolactin content of the pitu­
itary by all three measures of comparison over that of the
positive control (group 2) •

The electrically-stimulated rats

(group 3) also showed a marked increase in pituitary prolactin

60

content over that of both control groups, while the greatest
increase occurred in the pitocin-treated rats (Table 9).
These results show that suckling decreased pituitary
prolactin content, while electrical stimulation of the cervix
and pitocin injections apparently increased it.

61

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62

DISCUSSION
The results of Experiment I show that cortisone,
hydrocortisone and ACTH can induce mammary growth and secretion
in intact female rats.
of Selye

This Is in accord with similar findings

(I9?i].a, b) in ovariectomized, estrogen-primed rats.

Although the reports of Selye (l95iia, b) had not yet appeared
when this study was completed, these results confirm and ex­
tend his observations.

In addition they show that ACTH and

glucocorticoids can increase the prolactin content of the
pituitary and suggest that this mechanism is responsible for
the initiation of secretion.

In this study the greatest de­

gree of mammary growth and secretion were produced by corti­
sone rather than by ACTH, as reported by Selye (1952+a) •

The

latter employed almost 25 times as much ACTH as was used in
the present experiment.
The lesser degree of mammary growth noted with hydro­
cortisone as compared to cortisone in the present experiment
may

be due to over-dosage with the former, as indicated by

the considerable loss of body weight In these rats.
In Experiment II, cortisone injected alone into ovari­
ectomized rats induced some degree of mammary growth, but tnis
growth was more limited than in intact rats.

The alveoli were

y small and duct growth was only slightly increased over
that of the controls or estrone-primed rats.

63

Secretion in the cortisone—treated, rats was not as
narked as in the intact rats treated with cortisone in hxperi—
ment I*

When cortisone was injected into estrone— primed ratsj

alveolar growth and secretion were greatly increased over that y
of the rats treated with cortisone alone.

It appears there­

fore, that there is a synergistic action between cortisone and
estrone in ovariectomized rats.

It has been noted in the Re­

view of Literature that in normal mammary growth, estrogen may
produce a hyperaemia of the mammary stroma which could lead to
increased vascular permeability.

This in turn is believed to

lead to easy access to the gland of progesterone and other mam­
mary stimulating hormones

(Mixner and Turner, 19^3)*

This may

explain the increased alveolar growth seen in the ovariectomized
rats treated with both cortisone and estrone as compareo to those
treated with cortisone alone*
The explanation for the action of cortisone in inducing
mammary growth and secretion is not fully understood, but it
is believed that cortisone may exert a direct action on the
mammary gland as well as on the anterior pituitary.

Since

^

secretory activity was not prominent when cortisone or estrone
were given alone, it is apparent that the two hormones were
most effective when given together*

This is not surprising

since both have been shown to increase pituitary prolactin
content•
The glucocorticoids and ACTH appear to possess considerably less ability to increase pituitary prolactin content
in rats than estrogen, although they are as much or more potent

6i+

in this respect than testosterone or progesterone (Meites and
Turner, 1914-8)*
The adrenals are believed to be essential Tor main­
taining lactation in rats
Folley, 1914.7) •

(Nelson and Gaunt, 1937; Gowie and

However, the adrenal hormones have also been

reported to inhibit established lactation.

Flux et al (1951|)

and Shaw (195U- > 1955) reported a decrease in cows.

Meites and

Reineke (unpublished, 1955) found that 100 mg. of cortisone
injected daily into goats during the declining phase of lacta­
tion had no effect.

In Experiment III and IV, cortisone in­

creased the lactational response during the peak of lactation
(Cowiefs litter-growth index) and during the declining phase
of lactation (llth-l8th day post-partum) *

Although this in­

crease was only moderate, it was of the same magnitude as at­
tained with prolactin.

When these two hormones and growth

hormone were given in combination,
with prolactin or cortisone alone.

the response was the same as
Apparently there was no

synergistic action between the levels of cortisone, prolactin
and growth hormone used in this experiment.

It thus appears

that cortisone may stimulate or inhibit lactation, depending
on dosage and species used.
In Experiment IV the results indicate that both corti­
sone and prolactin retarded mammary involution when administered
to rats for 10 days after suckling had been terminated.

Hooker

and Williams (19^1) had previously shown that prolactin could
retard mammary involution In mice.

If cortisone acts on the

65

anterior pituitary to cause a release of prolactin, then its
effects in retarding mammary involution may be mediated, in
part at least, through this mechanism.

Since cortisone also

can induce mammary growth in the rat, it is probable that both
these actions account for its ability to retard mammary invo­
lution.
The inability of growth hormone to increase the lacta­
tional response or to retard mammary involution is difficult
to explain.

It is not in agreement with the positive results

of Cotes et. al. (l9l|-9),

Donker and Petersen (195l) and Shaw

(1955) in the cow, and of Meites and Relneke (1955) in the
goat.

In about IpO percent of the rats treated with growth

hormone, there were one or two abnormal young in each litter,
while the litters in the controls remained normal.

This sug­

gests that the growth hormone used may have been toxic, or
that it was detrimental when given to lactating as opposed to
non-lactating rats.
In Experiment IV there was a strong indication that
oxytocin and ACTH produced a galactopoietic effect.

While these

two hormones proved capable of increasing milk secretion during
the peak and declining phase of lactation, they did not have as
great an effect on the initial phase as cortisone or prolactin.
Also, despite their ability to increase lactation, ACTH and
oxytocin did not show any notable ability to retard mammary
involution after the young were removed from the dams.
difficult to explain in the case of ACTH.

This is

Presumably ACTH

66

stimulates adrenal cortical secretion, which in turn should
increase pituitary prolactin content and maintain mammary
growth.

It is possible however, that the dose of ACTH em­

ployed was suboptimal for both of these actions.

Closer exam­

ination of the histological sections indicate that there was
a more definite alveolar pattern in the ACTH-treated glands
than In the control glands.
The action of oxytocin in increasing litter weight
gains may have been directly on the myoepithelium of the al­
veoli, making available a greater supply of milk to the young
through its easier removal.

Benson and Folley (1956) were

able to markedly retard loss of secretory activity with intraperitoneal injections of 1 I. U. of oxytocin three times daily
given from the fourth to thirteenth day after parturition in
rats which had their young removed on the fourth day after par­
turition.

The present results are not entirely in agreement

with these workers.

However, It should be pointed out that

their studies were made on rats whose mammary glands were at
the height of activity (l|th-10th day), when involutionary
changes would be expected to be minimal, while the present
study was made on rats towards the end of lactation (18 days),
when a larger degree of involution might be expected.
Benson and Polley (1956) suggested that the effects of
oxytocin in retarding loss of secretory activity were not
mediated through a direct action on the mammary gland but
rather to a release of prolactin from the anterior pituitary.

67

lac tin has previously been shown to inhibit mammary involu­
tion in the mouse

(Hooker and Williams, 191+1) , and in this

study, in the rat.
The results in Experiment V show that in lactating rats,
suckling causes a release of prolactin, which confirms the
original report of Keece and Turner (1937).
have this effect.

Oxytocin does not

On the contrary these results suggest that

oxytocin in the lactating rat may increase the prolactin content
of the pituitary.

Meites and Turner 1191+8) have previously

reported that injections of posterior pituitary extracts do not
elicit a release of prolactin from the pituitary of rats,
guinea pigs and rabbits.

These results are therefore not in

agreement with the explanation of Benson and Folley (1956),
and suggest that another mechanism is responsible for the
favorable action of oxytocin in inhibiting decline in secretory
activity.

It is possible that oxytocin may cause the release

of other anterior pituitary hormones favorable to lactation,
such as ACTH.

Posterior pituitary extracts have been shown to

induce a release of ACTH (Saffran et al, 1955)* and it has
also been demonstrated that suckling induces a release of
ACTH (Gregoire, 191+6) .

It has not been demonstrated tnat oxy•*

tocin either stimulates or maintains mammary development.

J

68

SUMJ1ARY
1.

In Experiment I, physiological saline (0.85 percent)

or 2 mg. doses of ACTH, cortisone or hydrocortisone were in­
jected daily for 10 days into ip2 intact, mature female rats.
All three hormones produced growth of ducts, lobule-alveolar
development and secretory activity in the mammary glands.

This

was most marked for cortisone and least for ACTH at the dose
levels employed.
2.

Cortisone increased pituitary prolactin content by

about 23 percent, hydrocortisone by about Ip1 percent, and ACTH
by about 71 percent.
3*

In Experiment II, 100 mature female rats were

ovariectomized and divided into ten groups of 10 rats each,
according to weight.

Eight groups were divided Into two major

groups of IpO rats each (four groups of 10 each) and the re­
maining two groups (20 rats each) were used for controls.

The

treatment of the groups was as follows:
(1) controls,

saline only,

(2) controls, 1 mg. cortisone only.
The two major groups received either 5 ug. or 10 ug. of
estrone.

Within these two major groups cortisone was adminis­

tered to each sub-group as follows.
3) 2 mg. daily, ip) Ip tig. daily.
10 days.

1) none, 2) 1 mg. daily,

All injections were made for

69

il»

Cortisone alone stimulated lobule-alveolar develop­

ment and secretory activity in the mammary glands of tiie ovari—
ectomized rats.

However, mammary growth was not as pronounced

as observed in the intact rats of Experiment I.

On the other

hand, when cortisone and estrone were given together, marked
lobule-alveolar development was elicited ana secretory activity
was greatly increased.
3-

Cortisone augmented the pituitary prolactin content

of ovariectomized-estrone treated rats at the 1 mg. level.
However, when Ip mg • of cortisone was given daily with 10 u g .
of estrone, there was an inhibition of estrone action on the
pituitary.
6.

In Experiment III, the effects of cortisone on galac-

topoiesis was studied in

30

mature female rats.

These rats were

bred and at parturition their litters were reduced to 7 young
each.

The dams were injected daily auring an lb-day post­

partum period with 0.23, 0*3 or 1*0 mg. of cortisone.

The

lactational response was measured by the use of litter growth
rate.

The rats receiving 0.3 mg. cortisone showed a signifi­

cant increase In milk yield during the peak (6th-10tn day
post-partum) and during the declining phase of lactation (llth18

th day post-partum).
Cortisone at the 1.0 mg. level only slightly increased

the average litter growth during the
period.

1 6 —da^

experimental

However, during the declining phase of lactation,

70

significant increases in litter weight gain were noted, over
that of the controls,
6.

The results of Experiment III were confirmed in

Experiment IV,

In addition, Injections of cortisone were

continued for 10 days after the young were removed (ldth-2cith
day), in order to study its effects on mammary involution.

It

was found that cortisone at the 1.0 mg, level markedly retarded
mammary involution.

These mammary glands were comparable to

those of an untreated rat 5 days after removal of the young.
Cortisone at the 0.5 mg* level produced slightly less retarda­
tion of mammary disintegration, comparable to that of an un­
treated rat six days after removal of its litter.
7.

In Experiment IV the effect of growth hormone,

prolactin, oxytocin and ACTH on gaiactopoiesis and mammary in­
volution were studied.

Prolactin given at a dosage of 1 mg.

daily increased the average litter weight throughout the l8-day
post-partum period.

These increases were about equal to those

of the rats treated with 0.5 mg. of cortisone daily.

When

cortisone, prolactin and growth hormone were administered to­
gether, the response was of about the same magnitude as cor­
tisone or prolactin alone.

Thus no synergistic action on

galactopoiesls was exerted by these hormones.

Orcwth hormone,

when given alone, did not increase lactation.

8 . Prolactin (1 mg.) or prolactin, cortisone and
growth hormone given together, retarded mammary

Involution

comparable to that of a mammary gland of an untreated rat

71

5 days after removal of the young.

Growth hormone alone at

the level employed did not retard mammary involution.
9.

Oxytocin and ACTH exhibited galactopoietic effects

in parturient rats comparable to those of prolactin or
cortisone-treated rats.

However, the former two hormones

showed no ability to retard mammary involution following re­
moval of the young for 10 days.
10*

In Experiment V, suckling increased the pituitary

prolactin content in lactating rats.

Electrical stimulation

of the cervix of lactating rats appeared to increase pituitary
prolactin content over that of non-suckled or suckled rats.
Injections of oxytocin appeared to produce a large increase
in pituitary prolactin content over that of suckled, nonsuckled or electrically stimulated rats.

It appears,

there­

fore, that neither oxytocin nor electrical stimulation of the
cervix induces a release of prolactin from the pituitary.

72

BIBLIOGRAPHY
Azimov, G. J. and. Krouze, N. K. The lactogenic preparations
from the anterior pituitary and the increase of milk
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Balmain, J. H . , and Folley, S. J. In vitro effects of pro­
lactin and cortisone on the metabolism of rat mammary
tissue. Arch. Biochem. Biophys. 39 :168-191+, 1952,
Balmain, J. H,, Folley, S. J. and Glascock, R. F. Inhibition
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by cortisone in vitro and its antagonism by insulin.
Nature 169 :1+1+7-1+1+9, 1952.
Benson, G. K. and Folley, S. J. Oxytocin as stimulator for
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Bradbury, J. T. Study of enciocrine factors influencing
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Brownell, K. A., Lockwood, J. E., and Harman, F. A. A lactation
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Carr, J. L. Effect of Swingle's extract upon lactation in
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Clemenko, D. R., and hcChesney, E. W. Influence of epinephrine
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Cotes

G. W.
The hormonal control of lactation.
I. ^on-effect
*of the corpus luteum.
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P. M., Crichton, J. A., Folley, S. J. and foung, F. G.
* Galactupoietlc activity of purified anterior pituitary
growth hormone. Nature l 6 lj_:992-993, 191+9.

73

Cowie, A* T. The relative growth of the mammary gland in
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J.
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Gowie, A* T. Influence on the replacement value of some
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Cowie, A. T. and Folley, S. J. The measurement of lactational
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Cowie, A. T. and Folley, S. 0 • The role of the adrenal cortex
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40:274-285, 1947.
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12:15-28, 1995a.
Cross, B. A, Neurohormonal mechanisms of emotional inhioition
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Folley, S. J, and Young, F. G. Furtner experiments on the
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Gaunt, R. Adrenalectomy in the rat.
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Gaunt, R. Lactation in adrenalectomized rats.
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Gemzell, C. A.
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Gregoire C. Factors involved in maintaining involution of
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Hohn, E. D.
Estrogen response of mammary gland In adrenalectomized guinea pigs. Fed. Proc, 16:60, 1957.
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Hooker, C. W* and Williams, W. L. Retardation of mammary
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Johnston, R. F. and Smithcors, J. F. The effects of estrogen
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Lyons, W. R. The direct mammotrophic action of lactogenic
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Lyons, W. R. Lobule-alveolar mammary growth induced in hypophyseotomized rats by injections of ovarian hypophy­
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77

Meites

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R. P., Turner, C. W. and Hill, R. T.Mammary gland
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Endocrinology

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Walker, S. M. and Stanley, A. j. Induced lactation in virgin
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APPENDIX

82

Figures II4. to

23

are histological sections of mammary

glands from normal parturient rats showing daily involutionary
changes in mothers from the first to ninth day after removal
of their young•

In this series the young were removed

18

days

post-partum, and the mothers were sacrificed each day there­
after until the 27th day post-partum.
magnification of

130x

All figures are at a

and stained with hematoxylin and eosin.

Facing page 83

Figure

Eighteenth day post-par turn or immediately
after removal of young.
Sections are
comprised almost completely of large
lobules with dilated alveoli containing
copious secretion.
The nuclei lie at the
cell bases.
The thin connective tissue
septa contain greatly dilated, engorged
blood vessels*

83

Figure 14#

Facing page olp

Figure

If?.

Nineteenth hay post-partum or First
day after removal of young*
Sections
show alveoli with increased dilation,
and filled with secretion.
The epi­
thelium is flattened.
Other factors
are the same as on the previous day.

84

Figure 15*

Facing page

Figure 16.

8$

Twentieth day post-partum or second day
after removal of young.
The alveoli are
engorged with secretion and the epithelium
is slightly ragged.
In other areas the
alveoli are flllea with secretions,
Other
factors are the same as on the previous
two days.

85

Figure 16

Facing page 86

Figure 17.

Twenty-first day post-partuin or third
day after removal of young.
These
sections show alveoli and ducts markedly
engorged with secretions.
There is an
even greater flattening of the epithelium
with less secretion in the cells.

86

Figure 17*

Facing page 87

Figure

18.

Twenty-second day post-parturn or fourth
day after removal of young*
These sec­
tions show a little more flattening of
the epithelium and less cellular secre­
tion.
Some epithelial cells have lost
their nuclei, indicating early degenera­
tive changes.

87

Figure 18*

P a c in g

Figure 19.

page

88

Twenty-third day post-parturn or five
days after removal of young.
These
sections show that the lobules are small
and isolated, w i t h increased fibrosis.
The alveoli are rapidly degenerating and
are small.
Infiltration of fat has
increased.
Inspissated secretions are
found in the degenerating alveoli.
The
ducts are lined by cuboidal cells and
nuclei are ovoid.

88

Wimm

Figure 19*

Facing page 89

Figure 20.

Twenty-fourth day post-partum or six
days after removal of young.
These
sections show a more pronounced fibrosis
in the lobules with further degeneration
of the alveoli.
There are still a few
actively secreting alveoli.
The ducts
are filled with secretion.

89

r*ti
4*‘U

/

Figure 20.

F a c in g

Figure 21.

page

90

Twenty-fifth day post-partum or seventh
day after removal of young.
These sec­
tions show a large increase in fat.
The lobules are very small and an oc­
casional alveolus contains inspissated
secretion.
Little secretory material
is found in the aucts and the ductal
epithelium has about reached its m a x i ­
mum height.

Figure 21.

fa c in g

Figure 2?,

page

91

Twenty-sixth ctay post-parturn or eighth
day after removal of young*
These
sections show no pronounced change
from the previous day,
Farly lympho­
cytic infiltration can be seen.

91

m m r-

Figure 22•

Facing page 92

Figure 23 •

Twenty-seventh day post-partum or ninth
day ai'ter removal of youngThese sec­
tions show a large amount of glandular
degeneration.
The lobules are very small
and fibrotic.
Lymphocytic infiltration
is scattered throughout tne lobules.

92

Figure 23*