STUDIES OF THE ANTAGONISM BETWEEN PROLACTIN
AND THE OVARIAN HORMONES ON THE
INITIATION OF LACTATION

By
James Thomas Sgouris

AN ABSTRACT
Submitted to the School of 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
Year

Approved by

1955

ABSTRACT
1.

In Experiment I prolactin was incubated in vitro

for 1 hour at 58° C. with the following tissues:

liver,

kidney, brain and muscle of rats; mammary gland from lactating and ncnrlactating guinea pigs and rabbits; pigeon crop
glands; corpora lutea and the remaining ovarian tissue from
pseudopregnant rabbits.

After incubation the prolactin was

assayed in pigeons and directly compared with an equivalent
amount of prolactin which had been incubated in a tissuefree solution.
On a fresh tissue basis, mammary gland, pigeon crop
gland and the two ovarian tissues showed the greatest capacity
to inactivate prolactin.

Lactating mammary slices appeared

to be more effective in this respect than nonlactating mammary
slices.

Liver and kidney inactivated only about half as much

prolactin as the preceding tissues, while muscle and brain had
no effect on the prolactin.

Liver and kidney homogenates

were more effective in removing prolactin than slices of
these tissues.

When any of the tissues were boiled for 10

minutes prior to incubation with prolactin, they lost their
ability to inactivate the hormone.
2.

In Experiment II the inguinal mammary glands were

removed from 1? rats on the 12-15th days of gestation and
from 19 rats on the 4th day postpartum.

The glands were

homogenized and 100-mg.

samples were incubated with 0.2 mg.

of Squibb prolactin (20-25 I.U./mg.) at 38
hour.

±

0 .1° 0 . for 1

For controls, the same amount of prolactin was simi­

larly incubated without mammary tissue or with boiled
mammary tissue.

After incubation, the tissues were removed

and assayed in White Carne&u pigeons for prolactin activity.
The mammary homogenate from the lactating rats inacti­
vated 65.6 per cent of the prolactin compared with 19.5 per
cent for the tissue from the pregnant rats.

When corrected

for milk content, by lactose determinations, the former
tissue showed 8 times as much capacity to remove prolactin
as the latter.

These data suggest that the mammary glands

of pregnant rats cannot utilize prolactin to any marked
extent, and that this contributes to the absence of copious
lactation during gestation.
3.

In Experiment III a total of 63 albino rabbits of

both sexes, intact and castrate, were injected with 960
I.U. of estrone and 1 I.U. of progesterone daily for 25
days in order to induce optimal mammary development.

From

the 26th to 35th days, all except three rabbits were in­
jected daily with 2 mg. of prolactin (20-25 I.U./ mg.),
with or without one or both of the steroid hormones.

At

the end of the 35th day, the rabbits were killed and the
mammary glands were exposed and rated for intensity of lac­
tation.

The extent of mammary growth induced by these treat­

ments was determined by injecting radioactive phosphorus

(p32) 4 hours prior to sacrifice of the rabbits, and then
making radioautographs of the mammary glands.
The rabbits given prolactin and both steroids during
the last 10 days of the 35 day experimental period had prac­
tically no milk in their mammary glands (average rating of
less than l); those injected with prolactin alone had mammary
glands filled with milk (average rating of 3 to 4); those
given prolactin and progesterone showed no inhibition in
lactational response (average rating of 3 to 4); and the
rabbits injected with prolactin and estrone showed only a
slight decrease in milk secretion (average rating of 3 to
3).

The radioautographs indicated the presence of intensive

mammary growth in the rabbits treated with both steroids
throughout the entire 35 day period but not in the rabbits
given both steroids only for the first 35 days.

It is con­

cluded that in the doses employed, estrone and progesterone
together can effectively inhibit the milk-secreting action
of prolactin on the mammary gland of the rabbit, whereas

^

progesterone alone is ineffective and estrone alone is only
slightly effective in this respect.
4.

In Experiment IV 36 young rabbits were ovariecto-

mized, and after 10 to 14 days were injected daily with
0.96 mg. of estrone and 1 mg. of progesterone for 35 days to
induce optimal mammary development.

From the 26th to 35th

bays, 3 groups of 6 rabbits each were continued on the same
doses of steroid hormones together with 2, 4 and 8 mg. of
prolactin daily, respectively; 3 other groups of 6 each

were injected with 3 mg. of prolactin daily together with 0 ,
1/4 or 1/3 of the 3 steroid hormones used previously.

On

the 36th day the rabbits were killed and their mammary
glands were exposed and rated visually for lactational
response.
In the first 3 groups lactation was almost completely
inhibited when only 2 mg. of prolactin was injected daily in
the presence of the 3 steroid hormones, while 4 or 8 mg.
daily elicited good lactational responses.

In the second 3

groups, the greatest amount of milk was secreted when 2 mg.
of prolactin was given daily without the steroid hormones,
while the same dose of prolactin in the presence of either
1/4 or 1/3 of the steroid hormones produced a smaller flow
of milk.
It is concluded that the antagonism between mammary
growth and lactation is relative, depending on the balance
between the levels of prolactin and the 2 ovarian hormones
in the body.

When the mammary growth stimulus exerted by the

2 ovarian hormones is greater than the lactational stimulus
of prolactin, milk flow will be inhibited, and vice versa.
The application of these findings to explain the absence of
lactation during a non-lactational pregnancy or the simul­
taneous coexistence of lactation and pregnancy is discussed.
5.

In Experiment V, extracts of mammary tissue taken

from parturient rats and rabbits were prepared and injected
subcutaneously into mature and lactating rats.

Thirty mature

rats were divided into three groups and were injected daily

for 10 days as follows:

group 1 received a normal saline
i

solution; group 3 received 1.5 ml. rat mammary extract daily;
group 3 received 1.0 ml. rabbit mammary extract daily.

At

the end of the experimental period the rats were killed and
their pituitaries were assayed for prolactin content.

The

rat extract increased pituitary prolactin level by 41.8 per
cent, while the rabbit extract decreased it by 27.7 per cent.
Sixteen additional rats on the 4th day of lactation
were isolated for 4 hours from their litters and divided
into two groups of 9 and 6 , respectively.

Group 1 received

1 ml. of normal saline, while group 3 received 1.5 ml. rat
mammary extract in a single injection.

At the end of 24 hours

they were killed and their pituitaries were assayed for pro­
lactin content.

The rat mammary extract elicited a 16.9 per

cent increase in prolactin content.
On the basis of these limited data it appears that
mammary extracts from parturient rats and rabbits do not
influence the pituitary prolactin content when injected into
mature and lactating female rats.

Further work is necessary

before a definite conclusion can be drawn regarding the
effects of mammary tissue on pituitary prolactin secretion.
6.

In Experiment VI, the in vitro effects of prolactin

on the QOg of mammary slices from lactating rats was deter­
mined.

Eighteen rats on the 4th, 5th, 8th, 9th, 10th, and

13th day of lactation were decapitated and their inguinal
mammary glands were removed.

QOg was determined on slices in

Warburg flasks containing acetate-glueose-phosphate buffer
solution in the absence or presence of prolactin.

Prolactin

was found to increase oxygen consumption, particularly in
the latter stages of lactation.

This suggests that the

mammary glands from parturient rats are susceptible to
prolactin stimulation.
7.

Lactation is thought to be held in abeyance during

pregnancy by two mechanisms:

(l) the growth stimulating

levels of estrogen and progesterone on the mammary gland,
render it refractory to prolactin stimulation;

(2 ) the

relatively low level of prolactin secretion is insufficient
to overcome the inhibitory effects of the 2 ovarian hormones
on the mammary gland.

At parturition, the high levels of

the two ovarian hormones are greatly reduced, permitting the
increased amounts of prolactin from the pituitary to ini­
tiate lactation.

Lactation can continue during a subsequent

pregnancy because (a) prolactin secretion is maintained at
a high level by the milking act and (b) estrogen and pro­
gesterone are not secreted in sufficient quantities, parti­
cularly during the first half of pregnancy, to overcome the
action of prolactin on the mammary gland.

Other hormones

may also influence the initiation and maintenance of lacta­
tion, but they are believed to play a secondary role to the
factors mentioned above.

STUDIES OF THE ANTAGONISM BETWEEN PROLACTIN
AND THE OVARIAN HORMONES ON THE
INITIATION OF LACTATION

ByJames Thomas Sgouris

A THESIS
Submitted to the School of 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
Year

1955

ProQuest Number: 10008688

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TABLE OF CONTENTS

Page
INTRODUCTION
REVIEW OF LITERATURE....................................

1

Factors Controlling Lactation during Pregnancy and
Following Parturition
1 . Nervous Influences............................
4
S. Fetus Inhibition Theory ......................
5
3 . The Role of the P l a c e n t a ....................
6
4. The role of Corpora Lutea orProgesterone . ,
8
9
5. Role of Estrogen..............................
6 . Role of Estrogen and P r o g e s t e r o n e .............. 13
13
7. Mammary Growth versus Secretion .............
8 . Meites and Turner Theory ofLactation . . . .
15
EXPERIMENTAL............................................... 18
Experiment I. Inactivation of Prolactin by Different
Body Tissues in v i t r o ................................ 18
Experiment II. Differential Inactivation of
Prolactin by Mammary Tissues from Pregnant and
Parturient R a t s ....................................... 24
Experiment III.
Can the Ovarian Hormones Inhibit the
Mammary Response to Prolactin?.
............... 31
Experiment IV. Effects of Altering the Balance
between Prolactin and Ovarian Hormones on the
Initiation of Lactation in Rabbits....................40
Experiment V. Effects of Injecting Mammary Tissue
from Parturient Animals on Pituitary Prolactin
Content of R a t s ....................................... 46
Experiment VI. Effects of Prolactin upon Oxygen
Consumption of Mammary Tissue of Rats during
L a c t a t i o n ............................................. 50
DISCUSSION..................................................54
S U M M A R Y .................................................... 67
BIBLIOGRAPHY...........................

358313

73

LIST OF TABLES
Table
1.
3.

page
Effects of Various Hat Tissues on Inactivation
of Prolactin in v i t r o ............................ 31
Effects of Mammary, Ovarian and Pigeon Crop
Tissues on Inactivation of Prolactin in vitro .

22

3.

Inactivation of Prolactin by Mammary Tissue from
Hats in Different Stages of P r e g n a n c y ........... 27

4.

Inactivation of Prolactin by Mammary Tissue from
Rats in Different Stages of L a c t a t i o n ........... 28

5.

Lactational Responses in Rabbits Receiving
Prolactin with or without Estrone and/or
Progesterone....................................... 34

S.

Effects of Different Levels of Prolactin, Estrone
and Progesterone on Lactational Response in
R a b b i t s ........................................... 43

7.

Effects of Mammary Tissue from Parturient Rats
and Rabbits on the Pituitary Prolactin Content
of Cycling and Lactating R a t s ................... 48

8.

Effects of Prolactin upon the QOg of Mammary
Glands of Rats during L a c t a t i o n ..................51

LIST OF FIGURES

Figure

Page

1.

Lactational Responses of Castrated Male Rabbits
Receiving Prolactin with or without Estrone
and P r o g e s t e r o n e .................................35

3.

Lactational Responses of Ovariectomized Female
Rabbits Receiving Prolactin with or without
Estrone and Progesterone .......................

37

3.

Radioautographs of Rabbit Mammary Glands
Receiving Prolactin with or without Growth
Stimulating Levels of Estrone and Pro­
gesterone......................................... 38

4.

Lactational Responses of Ovariectomized Rabbits
to Varying Levels of Estrone and Progesterone
with Varying Levels ofP r o l a c t i n .................43

Dedicated
to my beautiful wife Katherine
and son Tommy

ACKNOWLEDGEMENT

The author wishes to express his sincere gratitude to
Dr. J. Meites, Professor of the Department of Physiology
and Pharmacology, for his generous assistance and construc­
tive criticism throughout the course of this work and during
the preparation of the manuscript.

He also wishes to express

his appreciation to Dr. B. V. Alfredson, head of the Depart­
ment of Physiology and Pharmacology, for providing facilities
and laboratory space to carry on these experiments; and to
Dr. W. S. Lundahl, Department of Natural Science, for his
assistance in preparing the radioautographs.

The author

also wishes to thank Dr. E. P. Reineke, Dr. W. D. Collings
and Dr. L. F. Wolterink for their advice during the course
of this work.
Thanks are also due to Mrs. R. C. Ogle for her tech­
nical assistance, and Mr. John Monroe for his help in the
care of experimental animals.
The author wishes to thank Dr. R. W. Bates, formerly
with E. R. Squibb and Sons and now with the National Cancer
Institute, Washington, D. C. for supplying prolactin and
Dr. Jack Reed of Chemical Specialties Company, New York,
New York for the crystalline estrone and progesterone
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
Council for providing financial support to Dr. J. Meites
which enabled the author to carry out this work.
Ttie author is particularly obligated to the Department
of Physiology and Pharmacology for providing a fellowship
for the first two years of this work.

INTRODUCTION

The onset of lactation following parturition was formerly
thought to be a natural consequence of the completion of
mammary development.

However, during the last fifty years new

concepts involving endocrine control of the mammary gland have
arisen.

Thus it has been shown that the initiation of lacta­

tion is primarily under the control of the endocrine system.
Numerous theories attempting to explain the abaence
or relatively minor secretory activity in the mammary gland

J
during pregnancy and the initiation of copious lactation at
parturition have been postulated.

These theories fall into

two categories ' (a) inhibitory theories, according to which
the lack of lactation during pregnancy is attributed to the
presence of inhibitory agents while the initiation of lacta­
tion is believed due to the removal of these agents;

(b) stimu­

latory theories, primarily that of Meites and Turner (1943a,
b, c), according to which the absence of milk secretion during
gestation was attributed to low lactogenic hormone production.
Estrogen, a stimulating agent, was believed to be prevented
from increasing prolactin secretion during pregnancy by the
overriding influence of progesterone.

At the completion of

pregnancy, estrogen was believed to gain the upper hand and
stimulate prolactin secretion by the pituitary, resulting in
the initiation of lactation.

J

None of the theories postulated gave adequate con­
sideration to the possibility that mammary growth may be
antagonistic to the initiation of lactation or adequately

^

explained the ability of some animals to maintain lactation
and pregnancy simultaneously.
This thesis is an attempt to throw additional light
upon this classical problem by determining:

(l) the degree

of inactivation, if any, of the lactogenic hormone by the
mammary gland and other body tissues in vitro; (2 ) whether
a differential could actually be demonstrated between the
ability of mammary tissue from pregnant and parturient rats
to inactivate prolactin; (3 ) whether the ovarian hormones
(estrogen and progesterone), either individually or together
could render the mammary gland refractory to prolactin stimu
lation; (4 ) the effects of altering the balance between pro­
lactin and ovarian hormones on the initiation of lactation
in rabbits; (5) whether mammary tissue from parturient ani­
mals could alter the pituitary prolactin content of rats;
(6) the effects of prolactin upon respiratory activity of
mammary tissue of rats during lactation.

It is hoped that

the results of these experiments have helped clarify the
endocrine control of lactation during pregnancy and at
parturition, although additional work will be required
before this problem can be completely resolved.

REVIEW OF LITERATURE

("it is evident that in the mammary gland a transition
from a growth phase to a secretory phase occurs during the
second half of pregnancy, culminating in the initiation of
copious milk secretion at parturition.

Under normal condi­

tions the growth phase is essentially completed by the
middle or two-thirds of pregnancy (Turner, 1939), and lacta­
tion is possible at this time.

Thus, if pregnancy is termi­

nated after mid-pregnancy, it is usually followed by lacta­
tion.

However, normally full lactation does not set in

until after the completion of gestation.

There are a number

of theories that attempt to account for this lack of abundant
lactation during pregnancy.

The classical ideas held during

the early days of physiology was that the initiation of
lactation was a passive phenomenon following parturition,
due to the removal of inhibitory factors that maintained
the gravid reproductive system.

The idea that lactation was

a passive process fell into disrepute with the discovery by
Strieker and Gr&ter (1929) that anterior pituitary extracts
contained a lactogenic factor capable of initiating lacta­
tion in non-lactating animals.)

Some of the most important

theories dealing with the initiation of lactation prior to
and following the discovery of prolactin will be reviewed
here.

NERVOUS INFLUENCES

IFollowing

parturition most animals nurse their young.

Therefore, the possibility that the nursing stimulus may be
responsible for the postpartum milk: flow must be considered.
If the suckling stimulus is the primary factor involved in
eliciting copious milk secretion,

then one would expect to

be able to initiate lactation in animals during pregnancy
by stimulating the nipples.)
rats with no success.

Masson (1948) attempted this in

^However, Selye and McKeown (1934a, b)

found in non-pregnant rats and mice that it was possible to

J

cause the mammary gland to proliferate and actually lactate
through stimulating the nipples by actively suckling litters)
^Reece and Turner (193?) in the rat and Meites and
Turner (1943b) in the rabbit set up unique experiments in
which pregnant animals were placed in cages with floors of
wire mesh containing large openings.

/

With the completion

of pregnancy the newborn dropped through the openings so
that the mothers were not suckled.

Lactation occurred a n d

pituitary prolactin was increased as in normal animals.

One

must conclude, therefore, in view of these observations, that
suckling is not necessary for the initiation of lactation at
parturition.

Suckling is necessary, however, for the main­

tenance of postpartum lactatioi^ (Selye, Collip and Thomson,
f

1934a; Selye and McKeown, 1934a, b; Turner and Reineke, 1938;
Meites and Turner, 1948a, b).

5

FETUS INHIBITION THEORY
|The fetus undergoes a tremendous spurt in growth and
\

development, particularly during the last third of preg­
nancy,

This caused many to speculate that lactation is not

/

/

initiated during the latter half of pregnancy because the
fetus takes nutrients from the blood that would otherwise
serve as precursors of milk*)
Another version stated that the developing fetus elicited
an inhibitory influence upon the milk secreting alveolar
cells of the mammary gland (Hildebrandt, 1904).

This inhibi­

tion supposedly prevented the breakdown of the alveolar cells
during lactation.

It is known now that the alveolar cells

per se do not constitute the milk, but only manufacture it.
^This theory appeared to receive additional support in the
*

experiments of Freud and Wijsenbeck (1938), who transferred
the fetuses from the uterus into the abdominal cavit}' of rats
and noted the continued absence of lactation:
fetuses were removed, lactation ensued.^

when the

This type of experi­

ment subjects the animal to extremely abnormal conditions
and one might suspect disturbances to any normal process such
as lactation.
(The termination of pregnancy by caesarian section in
its later stages in the rat results in the onset of lactation
within thirty-six hours; however, if the pituitary is removed
when the uterus is empty, no milk secretion occurs (Collip
ei a l . 1933).

Nelson (1934) partially confirmed this finding

in an experiment in which he removed the ovaries and embryos

6

from female guinea pigs during the 35th to 45th days of
pregnancy, leaving the placenta intact.
u e d absence of lactation.

He found a contin­

However, lactation occurred after

the expulsion of the placenta, indicating that the placenta
may be the primary inhibitory factor involved.
Therefore, in view of these observations and the wellknown fact that lactation may persist in the presence of
/
developing fetuses in subsequent pregnancies one is led to
conclude that the fetus plays no role in the inhibition of
lact tion.)
THE ROLE OF THE PLACENTA

'Halban

(1905) observed that when the placenta was

retained following delivery in parturient women, lactation
did not occur during the period of retention.

He believed

that the placenta was responsible for mammary growth during
pregnancy, preventing milk secretion until it was expelled.
Frankl (1923) implanted placental tissue into rats near

y

term and observed that no secretion occurred following
delivery as long as the graft remained active.

Smith and

Smith (1933) observed that rabbits with retained living pla­
cental remnants did not lactate, and thought the inhibitory
substance was estrogen secreted by this retained tissue^
[There is other evidence, however, which does not favor
the placental inhibition theory.

Placentas are frequently

retained in cows and do not inhibit the onset of lactation
(Petersen, 1942).

Copious lactation may also be maintained

during subsequent pregnancies in some species, such as
oattle.

Successful transplantation of fetal placental

tissues of pregnant rats into their eyes had no effect upon
lactation following parturition (Litt, 1933).
conclusions were drawn by Selye §t al.
ments with mice.

Comparable

(1934a) in experi­

They implanted whole mouse placentas

daily for 8 days into 6 lactating mice and found no dele­
terious effects upon lactation.)
ROLE OF UTERINE DISTENSION
tremendous growth of the fetus during the last
third of pregnancy submits the uterus to an abnormal dis­
tension.

It was postulated that the absence of lactation

during this time may be due in some way to mechanical dis­
tension of the uterus

(Selye, 1934; Selye ej al. . 1934a).

They removed the young by caesarian section during preg­
nancy or on the day of parturition and distended the uteri
with paraffin.
lactation.

This resulted in an apparent inhibition of

Inability of this theory to meet the test of

repeatability by Bradbury (l94l) and Greene (1941) resulted
in its lack of acceptance.

The latter showed that the

apparent failure to lactate was due to the effects of the
operation, since following recovery in their animals they
observed lactation even when the uteri were filled with
paraffin.

Again the observation that pregnancy and lacta­

tion can occur simultaneously in several species may be
taken as evidence against this theory.)

8
the

role

( It

of

corpora

lutea

or progesterone

is well known that ovariectomy or removal of the

corpora lutea in most animals usually terminates pregnancy.
If this occurs in late pregnancy and is accompanied by abor­
tion, lactation is initiated (Masson, 1948).

Therefore, one

is led to suspect that the ovary or one of its component
parts may be a possible inhibiting agent.

Hammond (1917)

postulated that the corpora lutea of pregnancy inhibited
lactation.

He based this on the observation that removal of

the corpora lutea from rabbits on the 12th day of pregnancy
resulted in lactation.

Drummond-Robinson and Asdell (192 6)

found that removal of the corpora lutea in goats after mid­
pregnancy also resulted in initiation of lactation.

They

attributed a mammary growth stimulus to the corpora lutea.
It was believed that the corpus luteum degenerated, thus
removing the growth stimulus, the alveolar cells would begin
to secrete and lactation would set in.

The decline of milk

yield with the advance of a subsequent pregnancy was also
explained by the progressive increase of this mammary growth
substance during pregnancy.
With the isolation of progesterone as the active factor
from the corpus luteum, and the demonstration of its increase
during pregnancy (Bachman

a_l_. . 1940; Lyon, 1946), the

concept arose that progesterone may prevent the onset of
lactation.

Walker and Matthews (1949) found that daily

treatment of rats with progesterone begun prepartally (2.5

9
mg.) or postpartally (5 mg.) could neither prevent nor
inhibit lactation.

The criterion of lactation was the

growth rate of nursing young.

Other workers

(Anselmino

al_., 1936a, b; Folley and Kon, 1938) Folley, 1943; Barsantini
and Masson, 1947) found no suppression of established lacta­
tion in rats given either moderate or massive doses of pro­
gesterone.

Abarbanel (1941) also reported that large doses

of progesterone were ineffective in suppressing the initi­
ation or maintenance of lactation in the postpartum human.
Only Reece and Turner (193?) found some degree of depression
of established lactation.

They gave crude progestin to

ovariectomized lactating rats and found that milk secretion
was decreased in about one-half of the animals.

Thus most

workers are in general agreement that progesterone per se
does not inhibit the initiation or the maintenance of lacta­
tion. j
ROLE OF ESTROGEN
Beerstecher (1941, 1943) observed an increased level
of estrogen in the urine of rats and rabbits as pregnancy
progressed.

Turner et al.

(1930) and Levin (1945) observed

a maximum concentration of estrogen in the urine and feces
of cows at term.

Zondek (l9 3l) and Goldberger and Frank

(1942) found that this hormone increased in the urine and
blood of women during pregnancy.

This indicates that in

several species there is a rise in estrogen during preg­
nancy.

\The progressive increase in the estrogen titer

10

throughout pregnancy and its decrease at parturition sug­
gested that this hormone may play a role in the inhibition
of lactation during pregnancy.
(1947) found that estradiol at
daily doses of 10 meg. and 1 mg. caused a marked inhibition
of lactation in normal rats.X

Walker and Matthews (1949)

found that prepartal injection of estrone did not prevent
the initiation of lactation either in ovariectomized (25
meg.) or in intact (25 meg. and 100 meg.) rats, but did
produce a delayed depression in the intact animals. (Postpartal treatment with 200 meg. of estrone begun on the
second day of lactation did not inhibit established lactation
in ovariectomized rats while moderate daily doses (5 meg.
and 10 meg.) of estrone inhibited lactation in intact rats.
This inhibition did not appear until the 10th to 12th day
following the initiation of treatment.

Large doses of

estrone (l mg.) inhibited lactation in both intact and
ovariectomized animals, but this was accompanied by marked
loss in weight of the mother.\
\0ther workers (Beece and Turner, 1937; Folley and Kon,
1938; and Folley, 1941) have established that estrogen will
inhibit lactation in the rat and cow, but the amount of this
hormone required was much larger than would be present in a
normal pregnancy.

In addition, it was necessary to begin

treatment with estrogen early in order to establish this
inhibition. \

^ Nelson (1936) developed a lactation hypothesis attri­
buting an inhibitory role to estrogen.

His theory not only

involves a mammary growth-promoting role for estrogen, but
in addition, an inhibitory effect on lactogenic secretion by
the pituitary.

He postulated that during pregnancy the

ovarian hormones, primarily estrogen, induced the prolifera­
tive changes that occur in the mammary gland, but that
estrogen inhibited the actual secretion of milk.

This

inhibitory influence was thought to act in two ways (a) by
suppressing the seoretion or release of prolactin by the
pituitary,

(b) by directly inhibiting the mammary gland.

At parturition, when the ovarian hormones sharply declined,
the inhibitory agent was removed and the gland was suscep­
tible to stimulation by lactogenic hormone.

The basis for

this theory of Nelson (1936) rests on a number of observa­
tions:

(l) Lactation was inhibited in normal parturient

female guinea pigs by injections of estrone.

If prolactin

was given simultaneously with estrone, lactation occurred.
However, estrone dosage could be increased to the point where
large amounts of prolactin failed to stimulate lactation.
(2) The injection of purified lactogenic hormone into preg­
nant animals failed to induce lactation^

(3 ) Crude pro­

lactin preparations did induce lactation but only after
pregnancy was terminated by abortion.

(4) Extirpation of

the ovaries was not invariably followed by abortion.

Lacta­

tion occurred only sifter fetal expulsion, whether they were
aborted or were e^elled normally at term.

(5 ) Lactation did

12

not occur when the ovaries and embryos were removed and the
placents was left intact, but did occur following expulsion
of the placenta.
^he inhibitory role attributed to estrogen by Nelson
(1936) can be criticized when one considers the following:

(l)

all of the experiments were conducted in guinea pigs, a species
which reacts to estrogen differently from most other species.
Thus, estrogen induces complete mammary growth in this species
whereas estrogen and progesterone are both required in other
species for complete lobule-alveolar growth.)

(2 ) Although

estrogen increases during pregnancy, it does not exert many
of its characteristic actions:
estrus,

(a) the animals are not in

(b) uterine motility is absent or at a minimum,

(c)

there is no or little increase in pituitary weight in several
species,

(d) estrogen usually induces a decrease in body

weight in rats but the opposite occurs during pregnancy.
The absence of these estrogenic effects is believed to be
due to the predominance of progesterone during pregnancy.
(3 ^ Artificial induction and enhancement of lactation with
estrogen is possibLe in goats and cows.

(4) Meites and

Turner (1948a) have shown that estrogen increases rather
than decreases pituitary prolactin content in the rat,
guinea pig, and rabbit and increases prolactin in the blood
of rabbits.

(5) The inhibition of established lactation which

is observed with large doses of estrogen may be due in part to
the toxic effects that it exerts on body metabolism.

Meites

13
and Turner (1948b) noted a progressive loss in appetite a n d
a reduction of food and water intake in goats, while Walker
and Matthews (1949) reported a drastic decrease in body
weight in lactating rats given estrogens*.
ROLE OF ESTROGEN AND PROGESTERONE
jMasson (1948) observed that when estradiol was given in
combination with progesterone to spayed rats the hormones
inhibited established lactation when the ratio of estrogen
to progesterone was 1 to 1000.

The milk secreting activity

of the mammary gland was estimated from the growth curve
and survival of the young.

Walker and Matthews (1949) found

that simultaneous injections of estrone and progesterone
begun prepartally or postpartally in the rat did not prevent
the initiation of lactation, but inhibited established lacta­
tion after 10-12 days of treatment.

A marked proliferation

of the glands indicated that growth of the mammary parenchyma
may play a role in the inhibition of milk secret ion).
MAMMARY GROWTH VERSUS SECRETION
Since mammary growth is completed by the middle or twothirds of pregnancy in most animals, one would presume that
growth per se is not responsible for the inhibition of lacta­
tion after mid pregnancy.

(However, Loeb and Hesselberg (1917)

postulated that mammary growth and milk secretion are incompatable and therefore lactation is absent during pregnancy.
Using as a criterion the number of mitotic figures in the

14
mammary gland, they found in the guinea pig and rat

that

as secretion progressively increased, mitosis in the mammary
cells became more rare.
Reece et al.

This relationship was confirmed by

(1940) who observed that when established lacta­

tion was inhibited by estrogen, the number of mitotic figures
in the mammary gland of rats increased.

Nelson et al.

(1943)

postulated that since estrogen, testosterone and desoxycortisone all stimulate mammary growth and inhibit lactation, all
substances which stimulate mammary growth have an inhibitory
effect on lactation.

Hammond (1937) suggested that the

decrease in milk production about the fifth month of a sub­
sequent pregnancy in the cow was due to active growth of the
udder .N
Folley and Kon (1938) reported that testosterone pro­
moted mammary growth and inhibited lactation and postulated,
as did Nelson si al.

(1943), that the ability of certain

hormones to cause mammary growth is accompanied by an ability
to inhibit lactation.

However, Folley (1942) later found

that massive doses of progesterone and desoxycorticosterone
produced no inhibition of established lactation in rats,
which was in disagreement with their hypothesis.
Orhe ability to elicit growth and simultaneously initiate
lactation in the udder of goats by Folley (1941) and Mixner,
Meites and Turner (1944) and in the udder of a cow by Walker
and Stanley (1941) constitute additional evidence against
this theory.

Cells are constantly being replenished in the

various organs of the body without disturbance to the normal

15
function of the organ*

As a new cell is formed and developed

others carry out the duties of the organ.

The ability of the

mammary duct system to secrete milk (Turner, 1939) indicates
that lobule-alveolar growth is not essential for a limited
degree of lactation to occur.

The ability of prolactin to

stimulate alveolar cell developments in the mammary gland
(Lyons, 1942) indicates further that mammary growth and
lactation can go on simultaneously.

This has actually been

observed during normal lactation in rats by Jeffers (1935)
and by Kuramitsu and Loeb (l92l) who found a direct correla­
tion between amitotic proliferation and intensity of milk
secretion.)
MEITES AND TURNER THEORY OF LACTATION
( The majority of the theories explaining the onset of
lactation at parturition were based on the removal of prepartum inhibitory agents.

Meites and Turner (1942a, b, c)

postulated that stimulatory as well as inhibitory factors
are involved in the initiation of lactation.
is based on the following:

Their theory

(l) Pituitary prolactin content

remains low during pregnancy, begins to increase prior to
parturition and increases sharply following parturition.
It is maintained at a high level during lactation in most
animals.

(s) Only estrogens, of all the hormones tested,

can significantly increase the pituitary prolactin content.
Even in physiological doses, estrogens can increase the
pituitary prolactin content to lactational levels in the

16
rat, guinea pigs, a n d rabbits, and this is accompanied by
an increased blood prolactin level in the rabbit.

During

pregnancy, when estrogen and progesterone are secreted in
increasing quantities, progesterone is thought to override
estrogen, preventing it from stimulating prolactin secretion.
However, at parturition, the levels of these hormones de­
clines rapidly and estrogen is believed to become predom­
inant, as indicated by the appearance of estrogenic effects
at parturition or shortly thereafter, i.e., estrus 1-2 days
postpartum in the rabbit, rat, and mouse.

Estrogen is

believed to activate the pituitary to secrete prolactin in
sufficient amounts to initiate lactation.A
The primary criticisms of this theory as given by Folley
and Malpress (1948) are:

(l) An increased pituitary pro­

lactin content does not necessarily indicate an increased
secretion of prolactin.

However, Meites and Turner (1948a)

have shown a simultaneous increase in blood prolactin with
increased pituitary prolactin in the rabbit.

(2) The pigeon

assay involves a tissue of mesodermal origin while the mammary
gland is of ectodermal origin.

Therefore, the identity of

prolactin with the pigeon crop proliferating factors is
questioned.

(3) ^ased on experiments with hypophysectomized

animals in which they were unable to initiate lactation with
purified prolactin alone, Folley and Malpress

(1948) claimed

that prolactin is not the only hormone involved in the ini­
tiation of lactation.

However, Meites (1954) has pointed

out that in the normal intact animal, with developed mammary

17
glands, only an increase in prolactin is essential for the
initiation of lactation.

Only prolactin has been shown to

be capable of directly stimulating the mammary alveolar
cells to secrete milk (Lyons, 1942; Meites and Turner,
1947:.)* which also proves its identity with the pigeon crop
stimulation factor.

The other hormones are believed to have

secondary albeit important supporting roles in the initia­
tion and maintenance of l&ctationi
/

It can be seen that no theory has yet been evolved
which can completely explain the absence of lactation during
an uncomplicated pregnancy, the initiation of lactation at
parturition, or the existence of a simultaneous pregnancy
and lactation in the same animal.

In the Discussion at

the end of this thesis, an attempt will be made to coordi­
nate the various lactation theories with the experimental
results presented herein.

EXPERIMENTAL
EXPERIMENT I.

INACTIVATION OF PROLACTIN BY DIFFERENT
BODY TISSUES in vitro

Purpose:
Little is known of the fate of lactogenic hormone in
the body.

It has been shown that a number of anterior

pituitary hormones are concentrated or inactivated by their
respective target organs (Seidlin, 1940).

Since injected

prolactin rapidly disappears from the bloodstream and only a
small fraction can be accounted for in the urine (Meites,
1940), it may be withdrawn or inactivated by its target
organ or other tissues in the body.

This experiment was

planned to determine the degree of inactivation, if any,
of the lactogenic hormone by mammary gland and other body
tissues ill vitro.
Method:
The following tissues were tested for their ability to
inactivate prolactin:

liver, kidney, brain and muscle from

female rats; mammary glands from lactating and non-lactating
guinea pigs and rabbits; pigeon crop glands; corpora lutea
and the remaining ovarian tissue from pseudo-pregnant rabbits.
After killing the animals, the tissues were quickly removed
and placed in ice-cold Ringer-phosphate solution.

The corpora

IS
lutea were carefully shelled out from the luteinized ovaries.
It is improbable, however, that the ovaries were entirely
freed of luteal tissue.

Tissue slices were cut with a

Stadie-Riggs microtome or homogenized with a glass tube and
plunger.

For tissue controls, several of the tissues were

boiled for 10 minutes in order to destroy enzymatic activity.
Approximately 500 mg. duplicate samples of tissue were
placed in standard Warburg flasks with 1.9 ml. of Ringerphosphate solution and 0.1 ml. of a solution containing 0.2
mg. of purified prolactin (Squibb:

1 mg. = 2 2 . 5 I.U.).

For

prolactin controls, only prolactin and Ringer-phosphate solu­
tions were incubated together at least six times during the
course of the experiments.

All flasks were placed in a water

bath at a constant temperature of 38+0.1° 0. for a period of
1 hour, except in one experiment liver slices were incubated
for 2 hours.

After incubation, the solutions were removed

and the flasks were rinsed with 2 ml. of distilled water.

The

fluids were combined and diluted so that 1.0 ml. contained
0.005 mg. of the original prolactin.
The pigeon method of Reece and Turner (1937) was used
to assay the prolactin.

White Carneau pigeons were injected

intradermally over both crop sacs with 0.1 ml. volumes daily
for 4 days.

A prolactin control solution was always injected

over one crop sac and compared with an equal amount of
tissue-incubated prolactin injected over the other crop sac.
On the 5th day the pigeons were killed and the crop glands

20

were removed and rated visually for degree of proliferation.
Hall (1944) reported that a 20 per cent difference in potency
between two lactogenic preparations could be detected by this
method of direct comparison in the same pigeon.
Results:
The data on the rat tissues are presented in Table 1.
It can be seen that when liver slices and prolactin were
incubated for 2 hours, 67 per cent of the prolactin was
inactivated, while when incubated for 1 hour, only 43 per
cent was inactivated.

Liver homogenate inactivated 70 per

cent of the prolactin in 1 hour, indicating that the homo­
genate was more effective in this respect than the liver
slices.

Kidney slices removed 46 per cent and kidney homo­

genate 76 per cent of the prolactin activity, again demon­
strating a greater inactivation by the homogenate.

When

liver or kidney slices were boiled, they lost their ability
to destroy lactogenic hormone.

Neither brain nor muscle

slices showed any significant capacity to inactivate pro­
lactin.
Table 2 shows the results of incubating prolactin with
mammary, crop gland and ovarian tissues.

Lactating mammary

slices or homogenate from guinea pigs removed all of the
lactogenic activity, while boiled mammary slices were com­
pletely ineffective.

Mammary slices from lactating rabbits

showed somewhat more ability to inactivate prolactin than
mammary slices from non-lactating, pseudopregnant rabbits.

21

TABLE 1.

EFFECTS OF VARIOUS RAT TISSUES ON INACTIVATION
OF PROLACTIN in vitro

Wt. of
tissue,
mg.

Treat­
ment

Liver slices^
Control^-

500

Fresh

5
5

0.55
1.65

67

Liver slices
Control

489

Fresh

5
5

1.05
1.85

43

Liver homog.
Control

481

Fresh

5
5

0.55
1.85

70

Kidney slices
Control

498

Fresh

5
5

1.00
1.85

46

Kidney homog.
Control

480

Fresh

3
3

0.41
1.75

76

Liver slices
Control

498

Boiled

5
5

1.90
2.00

.5

Kidney slices
Control

490

Boiled

5
5

1.20f
1.25

4

Brain slices
Control

515

Fresh

4
4

1.44
1.69

Muscle slices
Control

499

Fresh

4
4

0.81s
0.75s

Tissue

■^Incubation time was 2 hours.
^Immature pigeons.

Assay results
too.
Av.
Inacti­
pigeons/ rating/ vation,
1o
assay
pigeon

15
0

pp

TABLE 2,

EFFECTS OF MAMMARY, OVARIAN AND PIGEON CROP

TISSUES ON INACTIVATION OF PROLACTIN in vitro

Tissue

w t . of
tissue,
mg.

Treat­
ment

Assay results
No.
Av.
Inacti­
pigeons/ rating/ vation,
%
assay
pigeon
*

Lact. mam.
slices (g. Pig)
Control

4541

Fresh

5
5

0.15
1.85

92

Lact. mam.
homog. (g. Pig)
Control

4851

Fresh

5
5

0.00
3.30

100

Lact. mam.
slices (g. Pig)
Control

45311

Boiled

5
5

1.95
2.01

3

Lact. mam.
slices (rabbit)
Control

5081

Fresh

4
4

0.35f
1.50s

84

Nonlact. mam.
slices (rabbit)
Control

489

Fresh

4
4

0.44s
1.38s

68

Corpora lutea,
homog. (rabbit)
Control

330

Fresh

4
4

0.00
1.75

100

Other ovarian
tissue, homog.
(rabbit)
Control

319

Fresh

4
4

0.00
1.63

100

Crop slices
(pigeon)
Control

460

Fresh

5
5

0.15s
1.05s

86

Crop homog.
(pigeon)
Control

435

Fresh

5
5

0.00s
1.25

100

Crop slices
0
5
1.40s
Boiled
407
(pigeon)
1.40s
5
Control
^Since considerable milk was present in these tissues, their
true weights were much less than indicated here.
2 Immature pigeons.

23

Since the lactating mammary slices contained considerable
quantities of milk, there was actually more inactivation per
unit of tissue than indicated in the data.

Homogenates of

corpora lutea or of the reamining ovarian tissue from
pseudopregnant rabbits completely destroyed the prolactin.
Pigeon crop slices and homogenates inactivated 86 and 100
per cent, respectively, of the prolactin, while boiled crop
slices showed no capacity to inactivate the hormone.
Conclusion;
In general these data indicate that, when computed on
a fresh tissue basis, mammary, crop gland and ovarian tissues
were outstanding in their ability to inactivate prolactin.

Homo­

genates of these tissues appeared to possess at least twice the
ability of liver or kidney slices to inactivate lactogenic
hormone.

Brain or muscle slices showed little or no capacity

to remove prolactin, indicating that these tissues probably
do not normally metabolize prolactin in the body.
The importance of tissue enzymes in the inactivation
of prolactin is indicated by two observations.

First,

liver and kidney homogenates were more active than slices
of these tissues.

It seems reasonable to explain this on

the basis that more of the enzymes were in direct contact
with prolactin in the homogenates than in the tissue slices.
Second, when the tissues were boiled, thus inactivating-the
enzymes, the tissues were no longer able to alter prolactin
activity.

.

EXPERIMENT II.

24

DIFFERENTIAL INACTIVATION OF PROLACTIN BY
MAMMARY TISSUES FROM PREGNANT AND PARTURIENT
RATS

Purpose:
In experiment I there was some indication that mammary
tissue from lactating guinea pigs and rabbits had more
ability to inactivate prolactin than similar tissues from
pregnant guinea pigs and rabbits.

There is other evidence

that mammary tissue from lactating animals is more reactive
to prolactin than tissue from pregnant animals.

Thus

Folley (1952) has shown that prolactin can increase respira­
tory activity only in lactating mammary tissue of rats and
not in mammary tissue of pregnant rats.

Evidence will also

be presented in Experiment III demonstrating that the
ovarian hormones, which are secreted in large quantities
during pregnancy, are antagonistic to the action of pro­
lactin on the mammary glands.

It was the purpose of this

experiment to determine whether a differential actually
could be demonstrated between the ability of mammary tissue
from pregnant and parturient rats to inactivate prolactin.
Methods :
The inguinal mammary glands were removed immediately
after killing rats on the 12th to 15th, 15th to 17th, and
17th to 19th days of gestation, and from parturient rats on
the 1st, 4th, 15th and 30th days of lactation.

The tissues

35
were placed in ice-cold Ringer-phosphate solution, and homo­
genates were prepared with a motor-driven, ground-glass
homogenizer immersed in an ice-water bath.

Duplicate 100 mg.

samples of tissue were placed in standard Warburg flasks
containing 0.2 mg. of purified prolactin.

As a control for

each experiment, the same amount of prolactin and buffer were
placed in Warburg flasks without any mammary homogenate.

Each

flask, containing a total volume of 3.0 ml., was placed in
a water bath maintained at a temperature of 38 +, 0.1° 0., for
a period of 1 hour.

Prolactin was also incubated with

boiled mammary homogenate, and with boiled and fresh milk
from the mammary glands of lactating rats.

After incuba­

tion, the mixtures were removed and the flasks were rinsed
with the buffer.

The fluids were combined and diluted so

that 0.10 ml. contained 0.005 mg. of the original prolactin.
The fluids were assayed for prolactin in White Carneau
pigeons by the sensitive intradermal method of Reece and
Turner (1937).

A prolactin control solution was always

injected over one crop sac and compared with an equal amount
of tissue-incubated prolactin, injected over the other crop
sac of the same bird.

It has been stated that this tech­

nique permits the detection of differences of 20 per cent
between two prolactin preparations (Hall, 1944).
In order to determine the actual amount of mammary
tissue used in each experiment, a correction was made for
milk content by determining the total lactose per 100 mg.
of fresh tissue (Shaffer and Somogyi, 1933).

A lactose

26
value of 2.7 per cent was used for the rat milk, a figure
slightly below that given by Folley and Greenbaum (1947).
This introduces some degree of error, particularly in cal­
culating the amount of milk present in the pregnant mammary
gland which is usually not normal in composition, but an
even greater error would have resulted if no correction had
been made.

This can be seen from the fact that 60 per cent

or more by weight of the fresh mammary tissue from lactating
rats was found to be milk and not tissue.
Results:
The pertinent data relating to the pregnant rats are
tabulated in Table 3.

The amount of prolactin inactivated

by tissues from rats pregnant between 12 and 15 days was
19.5 per cent; at 15 to 17 days of pregnancy, 11.5 per cent;
and at 17 to 19 days of pregnancy, 19.6 per cent.

The aver­

age inactivation during this period was 16.9 per cent or
0.0338 mg. of the original 0.3 mg. of prolactin.

When cor­

rected for milk content, it wa3 calculated that 100 mg. of
mammary tissue from pregnant rats could inactivate 0.0433
mg. of prolactin.
The pertinent data relating to lactating rats are
tabulated in Table 4.

Lactating tissue inactivated 55.3

per cent on the 1st day postpartum, 65.7 per cent on the
4th postpartum day, 64.8 per cent on the 15th postpartum day
and 70.7 per cent on the 30th day of lactation.

The average

inactivation during this period was 64.1 per cent or 0.1383

37

TABLE 3 .

INACTIVATION OF PROLACTIN BY MAMMARY TISSUE FROM
RATS IN DIFFERENT STAGES OF PREGNANCY

Assay Results______
No.
No.
. Av.
rats/ pigeons/ rating/ pigeon
assay
assay
Control Experi_________________________mental

A m t . of
Calculated amt.
proProof prolactin
lactin lactin inactivated by
inacti- inacti100 mg. of
vated
vated actual tissue*
_______ £__________ rcg-

12-15th Day of Pregnancy
2
2
3
2
2
1
2
2
1
•

>
<1

5
5
5
5
5
5
5
5
5

1.08
1.08
.95
1.05
.89
.92
.95
1.25
1.10

.80
.90
.87
.80
.76
.90
.85
.90
.80

.0518
.0334
.0168
.0476
.0293
.0044
.1210
.0560
.0546

25.9
16.7
8.4
23.8
14.6
2.3
10.5
28.0
27.3

.0648
.0418
.0210
.1595
.0365
.0055
.0362
.0700
.0683

1.05

'.84

.0350

19.5

.0437

15- 17th Da
2
2
1
1
1
1

5
5
5
5
5
10

Av.

Av.

5
5
5
5
5
5

.0066
.0504

33.8
14.6
3.3
none
3.3
25.2

.0595
.0365
.0055
—
.0082
.0630

.0230

11.5

.0387

1.05
.89
.92
.54
.90
.91

.80
.76
.90
.60
.87
.68

.0476
.0293
.0044

.87

.77

17->19th
1
1
1
1
2
1

of Pregnancy

Be

of Pregnancy

.86
1.01
.73
.65
1.05
1.10

.88
.65
.73
.48
.70
.85

.0522
.0666
.0454

none
35.6
none
26.1
33.3
22.7

.90

.72

.0383

19.6

♦Corrected for milk content.

— —

.0712
—

—

.0890
—

.0652
.0832
.0567
.0490

TABLE 4.

INACTIVATION OF PROLACTIN BY MAMMARY TISSUE

FROM RATS IN DIFFERENT STAGES OF LACTATION
Assa y Results
A m t . of
Pro- Calculated amt.
No.
No.
Av.
pro- lactin
of prolactin
rats/ pigeons/ ratine:/pigeon lactin inacti- inactivated by
assay assay
inacti- vated
100 mg. of
Control Experi­ vated
actual tissue*
%
mental
mg.
mg.
1st Day of Lactation
3
2

5
10

Av.

.90
1.23

.35
.63

.1223
.0992

61.1
49.6

.3141
.2550

1.07

.49

.1107

55.3

.2845

4th Day of Lactation
2
2
1
2
3
2
3
1
2
2

5
5
5
5
5
5
5
5
5
5

Av.

1.30
1.05
1.55
.75
1.08
1.05
1.20
1.40
1.25
1.09

.35
.35
.45
.11
•44
.50
.55
.68
.45
.21

.1584
.1334
.1420
.1706
.1186
.1048
.1084
.1028
.1380
.1614

79.2
66.7
71.0
85.3
59.3
52.4
54.2
51.4
64.0
80.7

.4072
.3429
.3650
.4385
.3049
.2694
.2786
.2643
.3290
.4149

1.16

.40

.1338

65.7

.3413

15th Day of Lactation
3
2
1

10
5
5

Av.

1.00
.85'
1.00

.31
.25
.45

.1380
.1410
.1100

69.0
70.5
55.0

.3547
.3624
.2837

.95

.37

.1297

64.8

.3333

30th Day of Lactation
2
2
2
4
4
Av.

5
5
10
10
10

.80
1.00
1.06
.96
1.11

.19
.10
.44
.37
.36

.1524
.1800
.1168
.1228
.1353

76.3
90.0
58.4
61.4
67.6

.3917
.4627
.3002
.3156
.3475

.99

.29

.1414

70.7

.3634

♦corrected for milk content

29

mg. of prolactin.

When corrected for milk content the tissue

from lactating rats inactivated 0.3300 mg. of prolactin.
This is about an 8 fold difference in favor of the mammary
tissue from lactating rats.
No inactivation of prolactin occurred when it was incu­
bated with boiled mammary tissue or boiled milk.

Several

samples of fresh milk obtained from lactating rats inacti­
vated about 20 per cent of the prolactin.

However, this

does not necessarily prove that rat milk per se can destroy
prolactin, since the milk was obtained from cut slices of
mammary gland and undoubtedly contained some mammary tissue
exudates.

It should also be recalled that by the prolactin

assay method employed, a 20 per cent difference is of the
lowest level of significance.
Conclusions:
These data demonstrate that mammary tissues from partu­
rient rats have much greater capacity to inactivate pro­
lactin than similar tissue from pregnant rats.

The amount

inactivated by tissues from pregnant rats averaged 16.9
per cent (ll.5 per cent to 19.6 per cent range), while
lactating tissue inactivated about 64.1 per cent (55.3 per
cent to 70.7 per cent range).

When corrected for milk con­

tent it was found that lactating tissue could inactivate
about 8 times as much prolactin as an equal amount of tissue
from pregnant rats.

These data suggest that the mammary

glands of pregnant rats cannot utilize prolactin to any

30
marked extent, and that this probably contributes to the
absence of copious lactation during gestation.

31
EXPERIMENT III.

CAN THE OVARIAN HORMONES INHIBIT THE MAMMARY
RESPONSE TO PROLACTIN?

P urpose:
It is well known that the ovarian hormones during preg­
nancy prepare the mammary gland for lactation.

Mammary

growth in most animals is completed by mid to two-thirds of
pregnancy, as shown by the ability of the gland to secrete
milk in large quantities if pregnancy is terminated after
this time (Turner, 1939).

The continued presence of the

ovarian hormones at increasing levels during the latter half
of pregnancy leads one to suspect that they may play an equally
important role in inhibiting lactation during this period.
Although there is some basis for the belief that mam­
mary growth and lactation are antagonistic to each other
(Loeb and Hesselberg, 1917; Nelson, 1934, 1936), there is
also considerable evidence that mammary growth and lactation
can co-exist simultaneously in the same animal , as during a
simultaneous pregnancy and lactation or during the initia­
tion of lactation with ovarian hormones in cattle or goats
(see review of literature).

Therefore, it was the purpose of

this experiment to test whether the ovarian hormones, either
individually or together, could truly render the mammary
gland refractory to prolactin stimulation.
Methods *
A total of 63 young, previously unmated, New Zealand
White rabbits of both sexes were used in three experimental

33
trials.

The a n i m l s weighed from 5 to 7 pounds each at the

beginning of the experiments.

Intact males were used in the

first experiment, orchidectomized males in the second experi­
ment and ovariectomized females in the third experiment.

A

period of ten days was permitted to elapse after castration
in order to remove the influence of the gonads.

The animals

were fed commercial rabbit pellets, and both food and water
were available at all times.
All rabbits received daily subcutaneous injections for
the first 35 days of 960 I.U. of estrone and 1 I.U. of pro­
gesterone in separate 0.1 ml. volumes of corn oil.

Scharf

and Lyons (1941) had previously shown that this combination
elicited optimal mammary growth in male rabbits.

From the

3 6th to 35th day, all except three control rabbits received
3 mg. daily of prolactin by subcutaneous injection in 0.1
ml. of distilled water, together with one or both of the
steroid hormones.
In order to determine whether the mammary glands were
in a state of active growth during the last 10 days of each
experiment, several rabbits from each group were injected
intravenously with 50 microcuries of radioactive phosphorus
(P 33) per kg. of body weight, 4 hours prior to sacrifice.
Radioautographs were prepared of individual mammary glands
according to the technique outlined by Lundahl, Meites and
Wolterink (i960).
After killing the rabbits on the 36th day, the mammary
glands were exposed and rated visually for intensity of

33
lactation.

Ratings of 0 to 4 were assigned each rabbit

(Gardner and Turner, 1933).
Results:
The data for the 3 experiments are shown in Table 5.
In the first experiment, in which intact males were used,
the 3 control rabbits (group l) which received both steroid
hormones but no prolactin for the 35 days had no milk in
their mammary glands.

Groups 2 and 3 received both steroid

hormones for the full 35 days and prolactin from the 26th to
the 35th day.

No milk could be seen in their mammary glands

although an occasional drop could be squeezed from the
nipples.

Groups 4 and 5 were injected with the two ovarian

hormones for the first 25 days followed by prolactin alone
for the next 10 days.

Considerable lactation was evident

in all these rabbits (average rating of 3).

The mammary

glands of four representative rabbits from this experiment
are shown in Figure 1.
The results of the second experiment, performed on
orchidectomized rabbits, indicate that groups 1 and 2 respond­
ed the same as the similarly treated intact male rabbits of
the first experiment.

Group 3, which received prolactin and

estrone from the 26th to 35th days, had slightly less milk
in their mammary glands than the rabbits injected with pro­
lactin alone (group 2).

When progesterone was given together

with prolactin during the last 10 days of the 35-day experi­
mental period (group 4), no inhibition of lactation was
evident.

34

TABLE 5.

LACTATIONAL RESPONSES IN RABBITS RECEIVING

PROLACTIN WITH OR WITHOUT ESTRONE AND/OR PROGESTERONE

Exp.

Group
and No,
of
rabbits

Treatment
1st to
25th day

26th to
35th day

Av.
lactational
rating
per gland

)rabbits
i(3)
3(5)

Prog.3
Prog.3
Est.1 ♦

3(5)

E s t .1 *«■ Prog.3

4(5)
5(5)

Est.1
Est.1

Prog.3
Prog.2

0
Prolactin^ -*■
E s t . + P^og*
Prolactin*
Est. ♦
* ^rog.
J&st.
Prolactin^ only
Prolactin^ only

less than 1
less than 1
3
3

istrate male rabbits
l(5)
3(5)
3(5)
4(5)

Est.3- + Prog.3

Prolactin *
Est. + Prog.
Est.1 + Prog.3 Prolactin only
Est.1 •+ Prog.3 Prolactin + Est.
Estj.1
.1 t Prog.3 Prolactin + Prog.

le; ■s than
3
2
3

Castrate female rabbits
1(5)

Est.1
Prog." Prolactin
Prolactin +
E
st:.l + Prog.3
Est. * Prog.
Pro,
Est.1 r Prog.^
Prog.^ Prolactin only
o:
Est.1 +
Prog.3 Prolactin -► Est.
Est.1 -** Prog.3 Prolactin + Prog

1E s t . = estrone, 960 I.U. daily. ^
2Prog. = progesterone, 1 I.U. daily.
3Prolactin, 2.0 mg. daily.

less than 1
4
3
4

35

Figure 1.

All 4 male rabbits were injected with estrone

and progesterone for the first 25 days.

From the 26th to

35th day, the two rabbits on the left were injected with
prolactin, estrone and progesterone, and show no milk in
their mammary glands.

The two rabbits on the right were

injected with prolactin only during this period and show
considerable lactation.

36
The data, from the third experiment, on ov&riectomized
rabbits, are essentially in agreement with those of the
orchidectomized rabbits.

However, the degree of mammary

growth and quantity of milk secreted were greater (Figure 3)
than in the male rabbits.

Again estrone slightly inhibited

prolactin action (group 3) while progesterone did not inhibit
lactation (group 4).
Radioautographs of mammary glands from three intact
male rabbits of groups 3 and 4 of the first experiment are
shown in Figure 3.

It can be seen that mammary growth in

the two rabbits injected with both steroid hormones for the
full 35 days was active and widespread, as indicated by the
concentration of P^2 in the peripheral and nipple areas (the
regions of most active growth).

The mammary gland of the

rabbit from group 4, which received no steroid hormones from
days 26 to 35, shows growth stasis as indicated by the more
uniform distribution of P32 generally and a lesser concen­
tration in the peripheral areas.

The milk from this gland

showed only a minimal amount of radioactivity as compared to
that found in the mammary tissue, an observation in accord
with previous findings (hundahl

al. . 1950).

Conclusions *
These results are believed to demonstrate that when
estrone and progesterone are injected together in a ratio
previously shown to promote optimal mammary growth in male
rabbits, the mammary tissue is not responsive to prolactin

37

Figure 2.

Both ovariectomized rabbits received estrone and

progesterone for the first 25 days.

From the 36th to 35th

day, the rabbit above was injected with prolactin only and
its mammary glands are filled with milk.

The rabbit below

received prolactin, estrone and progesterone, and shows
complete inhibition of lactation.

38

Figure 3.

The two glands on the left are radioautographs

from two rabbits given estrone, progesterone and prolactin
from the 36th to 35th day.

The gland on the right is from

a rabbit given prolactin alone during this period.

Active

growth in the two glands on the left is indicated by the
concentration of

in the peripheral and nipple areas.

Growth stasis in the gland on the right is indicated by the
more general distribution of P ^ , with least in the peri­
pheral areas.

39
stimulation.

When either was injected alone, progesterone

produced no inhibition and estrone only a slight inhibition
of the mammary response to prolactin.

These data suggest

that the action of the two ovarian hormones on the mammary
gland during pregnancy render it refractory to prolactin
stimulation.

These results, therefore, at least partially

support the hypothesis that mammary growth and lactation are
antagonistic to each other.

To what extent they apply to

other species than the rabbit remains to be determined.

40
EXPERIMENT IV.

EFFECTS OF ALTERING THE BALANCE BETWEEN
PROLACTIN AND OVARIAN HORMONES ON THE INI­
TIATION OF LACTATION IN RABBITS

Purpose:
In Experiment III it was shown that a moderate dose of
prolactin could initiate lactation in rabbits with well
developed mammary glands.

This initiation of lactation could

be effectively inhibited if optimal, mammary growth-promoting
doses of estrone and progesterone were administered at the
same time.

In the absence of either one or both of the

ovarian hormones, this dose of prolactin elicited a good
degree of lactation.

The present experiment was undertaken

to determine the lactational response in rabbits to injec­
tions of (a) large doses of prolactin during treatment with
a constant but optimal combination of estrone and progeste­
rone for mammary development and (b) a moderate dose of pro­
lactin in the presence of less than optimal amounts of the
two steroid hormones.
Methods:
Thirty-six New Zealand White rabbits, weighing 5 to 6
pounds each, were ovariectomized and permitted 10 to 14
days recovery to remove the influence of the ovarian hor­
mones.

Each rabbit was then injected subcutaneously with

0.096 mg. of estrone and 1.0 mg. of progesterone daily in
separate 0.1 ml. volumes of corn oil for 35 days.

This com­

bination of the two hormones was shown by Scharf and Lyons

41

(1941) to elicit optimal mammary development in male rabbits,
and was used in the previous experiments.

From the 26th to

35th days, 3 groups of 6 rabbits each were continued on
the same doses of the 2 steroid hormones, together with 2,
4 or 8 mg. of prolactin daily, respectively; 3 other groups
of 6 each were injected with 2 mg. of prolactin daily together
with none, 1/4 or 1/2 of the amounts of the 2 steroid hor­
mones used previously.

The prolactin was injected subcutan-

eously in 0.1 ml. volumes of distilled water.

The rabbits

were killed on the 36th day and their mammary glands were
exposed and rated visually for lactational response, from
0 to 4 according to the Gardner and Turner (1933) method.
Commercial rabbit pellets and water were available at all
times to the animals.
Results •
The findings are summarized in Table 6.

The 2 mg. daily

dose of prolactin initiated an abundant lactation in the
absence of the two steroid hormones (group 1 and top animal
in figure 2) while the same dose of prolactin was almost
completely ineffective when injected together with 0.096 mg.
of estrone and 1 mg. of progesterone daily (group 2 and
animal number 3 in figure 4).

This is in agreement with the

previous observations in rabbits under identical treatment.
In the presence of the above amounts of the 2 steroids, 4 mg.
of prolactin daily elicited a moderate lactional response
(group 3 and animal number 4 in figure 4) while 8 mg. of

42

TABLE 6.

EFFECTS OF DIFFERENT LEVELS OF PROLACTIN,

ESTRONE AND PROGESTERONE ON LACTATIONAL
RESPONSE IN RABBITS
Group

Daily treatment
(26th-35th days
of experiment)

Av.
lactational
rating per gland

1

2 mg. Prolactin

2

2 mg. Prolactin «• .096 mg.
Est .1 + 1 mg. Prog.^

.2

4 mg. Prolactin ■* .098 mg.
Eat . + 1 mg. Prog.

1.9

8 mg. Prolactin + .096 mg.
Eat . + 1 mg. Prog.

3.2

2 mg. Prolactin -*■ .048 mg.
Est . + .5 mg. Prog.

1.1

2 mg. Prolactin + .024 mg.
Est . + .25 mg. Prog.

1.5

3
4
5.
6

lEst. m estrone.
^Prog. = progesterone.

4.0

43

Figure 4.

These five representative ovariectomized rabbits

received growth stimulating levels of estrone (0.096 mg.)
a n d progesterone

(l.O mg.) for the first 25 days.

26th to the 35th day they were treated as follows:

From the
the first

rabbit on the left was injected only with 3 mg. prolactin
daily and an abundant lactation was initiated (average rating
= 4.0).

The second animal from the left received 2 mg. pro­

lactin, 0.048 mg. of estrone and 0.5 mg. of progesterone; a
small amount of milk secretion was elicited (average rating
- l.l).

The third animal received 2 mg. prolactin daily,

0.096 mg. of estrone and 1.0 mg. of progesterone; practically
no lactation was initiated.

The fourth and fifth animals

received 0.096 mg. of estrone and 1.0 mg. of progesterone
and either 4 mg. or 8 mg. of prolactin,

respectively.

In

animal number four a moderate lactational response (average
rating “ 1.9) was elicited, while a good lactational response
(average rating = 3.3 was observed in animal number five.

prolactin daily (group 4 and animal number 5 in figure 4)
produced almost as much milk as in group 1.

When the 2

steroid hormones were injected in reduced amounts although
in the same ratio as used previously (group 5 and animal
number 2 in figure 4 ) the 2 mg. daily dose of prolactin was
sufficient to elicit at least a small amount of milk secre­
tion.

When the 2 steroids were further reduced, a much

greater amount of milk secretion was detected (group 6 and
animal number 1 in figure 4 )*

This was much greater in

group 2 but was less than in group 1 .
Conclusions:
These data demonstrate that (a) levels of estrogen and
progesterone which are optimal or near optimal for mammary
development in rabbits do not completely inhibit lactation
if a sufficiently large amount of prolactin is injected at
the same time, and (b) when the two steroid hormones are
injected in less than optimal amounts for mammary growth,
even a moderate dose of prolactin can elicit at least some
degree of lactation.

This indicates that the antagonism

between mammary growth and lactation is not absolute but is
relative, depending; on the balance between the levels of
prolactin and the two ovarian hormones in the body.

If the

balance is in favor of prolactin, lactation will be initiated,
while if the balance is in favor of the two ovarian hormones,
lactation will be inhibited.

43
EXPERIMENT V.

EFFECTS OF INJECTING MAMMARY TISSUE FROM
PARTURIENT ANIMALS ON PITUITARY PROLACTIN
CONTENT OF RATS

Purpose:
The level of the lactogenic hormone in the pituitaries
of most animals remains low during pregnancy, begins to increase
slightly prior to parturition and reaches its peak following
parturition (Meites and Turner, 1948a, b).

The latter have

also shown that estrogens are capable of increasing pituitary
prolactin content equivalent to postpartum levels in several
species.
It seemed possible that this increased prolactin level
following parturition might be mediated in part through the
mammary gland itself.

It was reasoned that if mammary tissue

from parturient animals could inactivate increased quantities
of prolactin and if it was more responsive to prolactin, it
might influence the pituitary to secrete more prolactin.

It

was the purpose of this study to determine whether postpartum
mammary tissue could alter the prolactin content of cycling
and lactating female rats.
Methods ‘
Mammary tissue 1 to 3 days postpartum from rats and from
parturient rabbits was collected separately and frozen solid.
When sufficient tissue 7/as accumulated, it was macerated in
a Waring blender with ice-cold normal saline.

The extraneous

tissue was removed by filtration through a Buchner funnel and

47

the filtrate was lyophilized.

One hundred mg. of dried

mammary extract from rabbits and rats was suspended in 1 ml.
1*5 ml. of normal saline respectively for injection.
Thirty mature female rats were divided into three groups
of ten each.
follows

•

They were injected subcutaneously, daily, as

(l) controls, 1 ml. normal saline; (2) 1.5 ml. rat

mammary extract daily;

(3 ) 1 ml. rabbit mammary extract daily.

At the end of 10 days of injections, the rats were sacrificed
and weighed.

The pituitaries were removed, weighed and

assayed in 30 pigeons according to the Reece-Turner (1937)
method.
Sixteen additional lactating rats (4 to 6 days post­
partum) were isolated from their litters for a period of 8
hours.

They were then divided into 2 groups of 9 and 7 each.

The larger group served as controls and received only saline
subcutaneously.

The other group was injected with 1.5 ml.

of rat mammary extract.
were killed and weighed.

Twenty-four hours later, both groups
The pituitaries were removed and

assayed for prolactin in 7 pigeons.
Results:
The findings are summarized in Table 7.

The rabbit

mammary extract appeared to decrease the pituitary prolactin
content in mature rats by 27.7 per cent after 10 days of
injection.

Though the site of injection in the rat was

varied every day, the area was highly inflamed in contrast
to the minimum reaction seen when rat mammary extracts were
used.

The rat mammary extract, in contrast to the raboit

48

TABLE 7.

EFFECTS OF MAMMARY TISSUE FROM PARTURIENT

RATS AND RABBITS ON THE PITUITARY PROLACTIN
CONTENT OF CYCLING AND LACTATING RATS

Animal

No.

Av.
Body

Treatment

wt.

__

gm-

Av. Av. Pigeon Units*
%
Pit. Per '“'Per"
Per
change
wt. Pit. mg.
100 gm.
mg.
pit. body w t .

Mature
rats

10

normal
saline

190.4 13.0 1.10 .0917

.578

Mature
rats

10

1.5 ml. rat 189.0 10. 5 1.56 .1488
mammary
extract

.826

+41.8

Mature
rats

10

1.0 ml.
rabbit
mammary
extract

190.3 10.9 0.85 .0779

.447

-37.7

Lactating
rats

9

normal
saline

350.5 14.6 1.60 .1096

.639

Lactating
rats

7

1.5 ml. rat 355.4 14.7 1.87 .1373
mammary
extract

.732

*Reece-Turner units.

—

+16.9

extract,

49
increased the pituitary prolactin content in mature

rats by 41.8 per cent after 10 days of injection.

An increase

of 16.9 per cent was also observed 24 hours after the rat
extract was given to rats on the 4th day of lactation.
Conclusions:
On the basis of these limited data, postpartum mammary
glands from rats and rabbits do not appear to influence
pituitary prolactin content in mature and lactating female
rats.

Further work is necessary before any definite con­

clusion can be drawn regarding the effects of mammary tissue
on pituitary prolactin secretion.

50
EXPERIMENT VI.

EFFECTS OF PROLACTIN UPON OXYGEN CONSUMPTION
OF MAMMARY TISSUE OF RATS DURING LACTATION

Purpose:
Kleiber el &1.

(1943) and Folley and French (1949) have

reported that the oxygen consumption of the lactating mammary
gland is considerably higher than that of the mammary tissue
in pregnant rats.

This increased metabolic activity in the

mammary gland following parturition is known to be accompanied
by a relatively high level of prolactin secretion by the
anterior pituitary.

Folley (1952) reported that the addi­

tion of prolactin in vitro to mammary tissue from parturient
rats increased net respiratory activity.

It was the object

of this experiment to determine whether the addition of pro­
lactin to lactating rat mammary tissue .in vitro would in­
fluence oxygen consumption.
Method:
Lactating mammary glands were obtained from 18 rats of
the Carworth strain.

On the 4th, 5th, 8th, 9th, 10th and

12th days after parturition the animals were killed by decapi­
tation.

The inguinal mammary glands were quickly removed and

rinsed in Ringer*s solution and placed on moist filter paper.
Slices of approximately 0.4 to 0.5 mm. in thickness were cut
with a Stadie-Riggs microtome.

Samples weighing approximately

200 mg. were placed in a Warburg flask containing 2.8 ml.
Ringer-phosphate buffer, 0.3 per cent glucose and 0.02 M
sodium acetate.

The center well contained a folded filter

paper wick and 0.1 ml. of 20 per cent KOH solution.

Duplicate

TABLE 8.

EFFECTS OF PROLACTIN UPON THE Q03 OF MAMMARY
GLANDS OF RATS DURING LACTATION

Days of
lactation

No. of
rats

QOg*

Q°3*
in presence
of prolactin

%

change

4

2

1.74

2.19

4

2

2.86

3.12

4

2

3.67

3.06

14.6)

5

2

1.67

2.24

34.1

8

2

3.78

5.63

49.0

9

2

2.20

3.44

9

3

2.78

3.71

56.3)
) 44.9
33.5)

10

2

2.98

2.95

13

2

3. 04

4.90

25.9)
9.1) 16.5

—
61.2

*QOg of dry tissue not corrected for milk content.

52
samples of mammary tissue with and without added prolactin
were used.

A total of 2 mg. of Squibb prolactin (30-25 I.U./

m g . ) were added to the former flask.

All incubations were

carried out at 37 +_ 0.1° C., in the presence of a gas mixture
of 95 per cent oxygen and 5 per cent carbon dioxide.

After

an equilibration period of 10 minutes, the flasks were agi­
tated for 2 to 3 hours and the oxygen consumption was deter­
mined at intervals of 15 minutes each.

Additional samples

of 100 to 200 mg. of tissue were weighed and dried for 48
hours at 100° C.

These were weighed again to determine the

percentage dry matter of the tissue.
ported in terms of QOg,
of dry tissue per hour.

The results are re­

the mm.® of oxygen consumed/mg.
The QOg values given are averages

based usually on the first eight 15-minute periods.

At the

end of each experiment the solutions had a milky-white color
indicating the presence of milk.

No correction was attempted

for the milk content of the tissues.
Results:
The results are tabulated in Table 8.

Oxygen consump­

tion during lactation was increased by the addition of 2 mg.
of prolactin in acetate-glucose-Ringer phosphate medium.
This increase appears to be lowest early in lactation, averag­
ing only 16.5 per cent in 6 animals on the 4th day of lacta­
tion.

On the 13th day the average increase was 61.2 per cent

in 2 animals.

The Q0g was relatively low compared to the

figures given by Folley and French (1949) and this is

53
undoubtedly due to the fact that no correction was made in
the present experiment for the milk present.

The results in

Experiment II indicate that the milk content of the mammary
gland during lactation accounts for about two-thirds of the
dry matter present.

This indicates that these QO^ values

would be increased approximately three-fold if corrected
for milk content, bringing them within the range of the
figures of Folley and French (1949).

However, the relative

increase in QOg would not be altered if this additional
correction was made.

Therefore, these results remain valid

as they are.
In one experiment involving animals on the 10th day of
lactation, prolactin had no effect on QOg.

In this case the

prolactin was added from the side arm of the flask rather
than being present in the original solution.

For some

unknown reason there was no increase in QOg when this pro­
cedure was used.

One may speculate that it may be a matter

of solubility, i.e., the prolactin did not go into solution
readily.

However, there was no progressive increase in QOg

as one would expect if the prolactin acted more slowly.
Conclusion:
When added in vitro, prolactin was capable of increasing
the QOg of lactating mammary tissue from rats.

This empha­

sizes the susceptibility of mammary glands from parturient
rats to prolactin stimulation.

Folley (1953) reported that

the in vitro addition of prolactin to mammary tissue from

pregnant rats had no effect on respiratory activity, pro­
viding further support for the view that prolactin and the
ovarian hormones are antagonistic in their actions on the
mammary gland.

DISCUSSION

MAMMARY GROWTH VERSUS LACTATION
(The continued presence

of lactation

during a subse­

quent pregnancy indicates that the ability of estrogen and
progesterone to render the mammary gland refractory to
prolactin stimulation is not absolute.

^

Rather it depends

on the balance between the amount of prolactin and the two
ovarian hormones, estrogen and progesterone, in the body,)
as shown in Experiments III and IV.

In the latter experi­

ment the level of prolactin was increased and the two steroids
were maintained at the same level, or the same level of pro­
lactin was maintained and the doses of the two steroids were
decreased.

In the first series, as the level of prolactin

was increased to 4 or 8 mg daily, a good lactational response
was observed in contrast to little or no milk secretion with
2 mg. of prolactin daily.

In the second series the greatest

amount of milk was observed in the mammary glands when the
two steroids were completely withdrawn and only 2 mg. of
prolactin daily was administered, while the same dose of
prolactin in the presence of either 1/2 or 1/4 of the two
steroid hormones produced a smaller flow of milk,

(in view

of these results, one may conclude that (l) the levels of
estrogen and progesterone which are optimal or near optimal
for mammary development in rabbits do not completely inhibit

56
lactation if sufficiently large doses of prolactin are injected
at the same time and (2 ) when the two steroid hormones are
injected in less than optimal amounts for mammary growth,
even a moderate dose of prolactin can elicit at least a
moderate degree of milk secretion.

This indicates that the

/

antagonism between mammary growth and lactation is relative,
depending on the balance between the levels of prolactin and
the two ovarian hormones in the body.

If the growth stimulus

exerted by the two ovarian hormones on the mammary glands is
greater than the lactational stimulus of prolactin, then
milk production will be inhibited, and vice versa.

This

does not exclude the possibility that other hormonal or nonhormonal factors may modify the lactational response)
(Desclin (1952) has provided further evidence that large
doses of prolactin can overcome the inhibitory action of
the ovarian hormones on mammary secretion.

He observed that

when 30 I.U. of prolactin were injected daily into mature
female rats, there was intense luetinization of the ovaries
but no mammary secretion; however, when 200 I.U. of pro­
lactin were injected daily, there was mammary secretion
despite intense luteinization of the ovaries.

Earlier,

Nelson (1934) had reported that lactation in parturient
guinea pigs was not suppressed by estrogens if a sufficient
dose of prolactin was injected at the same time^
(The initiation of a localized lactation in rabbits by
intraductal injections of 10-20 I.U. of purified prolactin
into a single nipple, without terminating pregnancy (Meites

j

57
and Turner, 1948a) is additional proof that prolactin can
overcome the inhibitory influence of the ovarian hormones
on the mammary tissue during pregnancy^

The failure of

Nelson (1936) a n d others to initiate lactation in pregnant
animals with crude prolactin preparations, without first
inducing abortion, may be due to contamination with other
pituitary hormones.
^It is believed that the absence of milk flow during
pregnancy or the presence of abundant lactation in animals
which are simultaneously pregnant and lactating, can be
accounted for on the basis of the balance present between
the levels of prolactin and the two ovarian hormones in the
body.

During a non-lactational pregnancy, the combined

action of estrogen a n d progesterone stimulates mammary
development but renders it relatively refractory to prolactin
stimulation.

In addition, the secretion rate of prolactin

by the pituitary during a non-lactational pregnancy is too
low to initiate abundant lactation (Meites and Turner, 1948a,
b).

Following parturition, prolactin secretion greatly

increases and at the same time the reduction in ovarian
hormones enhances the sensitivity of the mammary glands to
prolactin stimulation, serving to initiate copious milk flow.
Lactation can continue in parturient animals which subse­
quently become pregnant, probably because (a) the initial
level of prolactin secretion is already high and remains
high as a result of the continued milking stimulus (Reece

58

a n d Turner, 1937; Meites and Turner, 1943b) and (b) estrogen
an d progesterone are secreted at lower levels during early
than in late pregnancy, and hence they would not appreciably
antagonize prolactin action and decrease milk production
until the latter part of gestation.

Thus, in dairy cows

which are simultaneously lactating and pregnant, there is
no pronounced drop in milk yield until after the 5th month
of gestation, following which time the decline is about 20
per cent greater than in non-pregnant cows (Hammond, 1927;
Turner, 1939)].
It is clear from the present work that the theory that
mammary growth and lactation are antagonistic (Loeb and
Hesselberg, 1917; Nelson, 1936; Folley and Kon, 1937) is
not totally incorrect.

However, the present work indicates

that the original concept must be modified considerably.
/Mammary growth and lactation are antagonistic only when
optimal or near optimal amounts of the two ovarian hormones
are present.

Even then, a large amount of prolactin can

initiate some degree of milk secretion.

It is obvious,

therefore, that the antagonism between mammary growth and
lactation is not absolute but is relative.
THE ROLE OF THE OVARIAN HORMONES
It is clear from Experiment III that progesterone per
sje does not inhibit the initiation or maintenance of lactation.

This is in agreement with the observation of previous

workers, who found that even large doses of progesterone

j

could not inhibit the initiation or the maintenance of
established lactation (Walker and Matthews, 1949; Folley,
1943; Barsantini and Masson, 1947).
When only estrone was given in addition to prolaotin,
this resulted in only a slight inhibition of lactation.

This

is in general agreement with the observations of previous
workers, who found that much larger doses of estrogens are
required to inhibit established lactation in ovariectomized
as compared to intact rats (Masson, 1948; Folley and Kon,
1938; Anselmino and Hoffmann, 1936a, b).

There is evidence

that estrogen in large doses m y have certain toxic effects
rather than specifically inhibit lactation.

Edelmann and

Gaunt (1941), Trentin and Turner (1941), Walker and Matthews
(1949) found that large doses of estrogen caused lactating
rats to lose weight.

Meites and Turner (1948b) also found

that the depressing effects of estrogen on lactation in
goats is mediated largely through a loss of appetite^)
^ In the guinea pig where only estrogen is necessary for
complete mammary growth and for inhibiting lactation (Nelson,
1936, 1950), one must consider the possible effects of this
hormone on the adrenals.

It has been shown that small doses

of estrogens stimulate increased steroid hormone output by
the adrenal cortex of the guinea pig (Kimeldorf et al_.,
1947; Zondek et al.» 1952).

In view of the ability of the

adrenal cortical hormones, such as desoxycorticosterone,
cortisone and progesterone, to elicit mammary growth (Folley
and Kon, 193 8; Nelson

aA*, 1943; Selye, 1954; Johnson and

60

Meites, 1955), the* adrenal steroids must he considered when
estrogen is given to guinea pigs or to other species.

For

example, the inhibition of lactation with small daily doses
of estrogen (5 meg. daily) in intact rats and the observa­
tion that only simultaneous injections of progesterone and
estrone inhibit lactation in ovariectomized rats (Walker and
Matthews, 1949) indicates that estrogen per se is not respon­
sible for the inhibition of lactation but that this is proba­
bly due to a synergism between estrone and progesterone.

The

source of the progesterone-like steroid in the case of the
ovariectomized rats is probably the adrenal cortex^
^ The above indicates that the inhibition of established
lactation is due to a synergistic action of estrogen and
progesterone, and is in harmony with the results of Experi­
ment III where it was shown that only when the two steroids
were administered in doses which were optimal or near optimal
for promoting mammary growth, that the moderate dose of
prolactin used initiated little or no milk flow.
also in agreement with Desclin's

This is

(1953) observations.

He

found that when ovariectomized rats were injected daily with
0.1 mg. of estradiol benzoate, 4 mg. of progesterone and 30
1.U. of prolactin, there was excellent mammary growth but
no lactation.

However, when only progesterone was injected

with prolactin, or when only diethylstilbestrol was given
with prolactin, abundant milk secretion was initiated.

J

61

B OLE OF THE PIiACEUTA

The placental theory of inhibition is not entirely in­
correct in view of the endocrine functions attributed to it.
It appears that the placenta in some species gradually
assumes certain ovarian functions which allow gestation to
continue (Newton, 1938).^ It has been shown in the human
\

being, guinea pig, mare and monkey that ovariectomy at midpregnancy does not terminate pregnancy (Asdell, 1928;
Douglass, 1931; Probstner, 1931).

In the monkey and human

being ovariectomy at this time does not alter urinary
estrogen or pregnanediol levels (Turner, 1949).

This is

believed to be due to the ability of the placenta to act
as an endocrine gland and secrete these hormones (Haterius,
1936; Leonard, 1945; Newton, 1938; Selye

a l . . 1935).

Since placental tissue in the above species probably assumes
the role of the major producer of estrogen and progesterone
during the second half of pregnancy, it may play an important
role in inhibiting lactation during gestation.)
INACTIVATION OF PROLACTIN BY BODY TISSUES
The results of Experiment I on the inactivation of
prolactin by body tissues in vitro show that on a fresh
tissue basis, mammary, crop gland and the ovarian tissues
were outstanding in their ability to inactivate prolactin.
These tissues appeared to possess at least twice the abili­
ty of liver and kidney slices to inactivate this hormone.
This is in agreement with Seidlin (1940) who observed that

62
gonadotrophic and thyrotrophic hormones are withdrawn from
the blood stream by their respective target organs.

He

based this on the observation that thyroidectomized and
gonadectomized guinea pigs excreted considerably more of
the injected thyrotrophin and gonadatrophin, respectively,
than did intact animals.

In vitro

incubation studies of

these hormones, with their respective target organs, resulted
in considerable inactivation of these hormones after only
one hour.
Sonenberg (1951) reported that a radioactive prepara­
tion of prolactin labeled with I^3^* was concentrated in the
ovary, liver, adrenal and kidney, but found no significant
concentration of radioactivity in the mammary tissue of the
rat under various physiological conditions.

One may question

the potency of this preparation since its activity was deter­
mined in only two birds, a number much too small to give an
accurate indication of biologic activity.

In addition, the

control injections were made in different pigeons, providing
an additional source of error.

In contrast to these results,

Cox (1951) reported that 20 minutes after intravenous injec­
tion of I131 labeled prolactin, the highest concentration
was found in the liver and kidney, and appreciable amounts
were also present in mammary tissue, milk, ovaries, and
adrenals.

Her results indicated that prolactin promptly

left the circulation and underwent rapid breakdown.

The

result of the present study indicates that inactivation may

63

occur in numerous tissues but primarily in the mammary
gland and ovary.
Meites (1940) injected prolactin subcutaneously into
rabbits and followed its excretion in the urine.

He was

able to recover less than 1 per cent during the first 24
hours after injection, only a trace during the second 24
hours, and none during the third 24 hours.

When prolactin

was injected intravenously, none was recovered in the urine
during the 72-hour period which followed; and when attempts
were made to recover it from the bloodstream, about half
was found at 5 minutes and none at 1/2 hour and 1 hour
after injection.
The results of the first experiment suggested that the
ability of mammary tissue to inactivate prolactin may depend
on whether the animal is pregnant or lactating.

In the

second experiment it was found that lactating mammary tissue
taken from rats on the 4th day after parturition showed
about eight times as much ability to inactivate prolactin
as mammary tissue taken from rats on the 12th to 15th days
of pregnancy. ^ This suggests, in agreement with the results
of Experiments III and IV, that (l) mammary glands of preg­
nant rata are refractory to prolactin stimulation.

The

refractoriness is believed due to the action of the growth
stimulating levels of the two ovarian hormones, and (2 )
mammary glands of lactating rats are sensitive to the action
of prolactin because the refractory effect of the ovarian
hormones is not present.

The preceding suggests a possible

64

mechanism for the maintenance of prolactin at high levels
during lactation.

The gland is using and inactivating the

available pituitary prolactin, and it may be withdrawing it
from the circulatory system at a rate that exceeds the ability
of the pituitary to produce prolactin.

The pituitary, there­

fore in attempting to maintain its normal secretory level,
may be indirectly stimulated by the high metabolic activity
of the lactating mammary gland.

This possibility remains

to be tested.j
The possibility was also considered that the lactating
gland may furnish a chemical mediator which activates the
pituitary to secrete more prolactin (Experiment VI).
Extracts of postpartal lactating mammary glands were made
from rats and rabbits.

These were injected into mature and

lactating rats and their pituitaries were assayed for pro­
lactin.

The rabbit mammary extract caused a decrease in

pituitary prolactin content in mature rats, while the rat
mammary extract elicited an increase in pituitary prolactin
content in both mature and lactating rats.

The results

are therefore insufficient to allow one to draw any defi­
nite conclusions regarding the presence of a chemical or
hormonal mammary stimulator of pituitary prolactin secre­
tion.

This does not exclude the possibility that the

parturient mammary gland may provide a nervous stimulus to
pituitary prolactin secretion.

65

OXYGEN UPTAKE OF THE MAMMARY GLAND
In Experiment II it. was shown that the lactating gland
of parturient rats had more ability to inactivate prolactin
than the growing mammary gland from pregnant rats.

The post­

part al mammary gland is more susceptible to prolactin stimu­
lation, and apparently this is accompanied by an increase in
mammary metabolic activity.
true by Kleiber et al.

This has been shown to be

(1943) who conducted the first in

vitro studies on the respiration of mammary tissue of rats.
They found no difference between oxygen consumption on the
20th day of pregnancy and throughout lactation on a moist
tissue basis.

However, when calculated on the basis of dry

matter content, they found a progressive increase in
from 0.9 at the end of pregnancy to 2.9 on the 21st day
after parturition.

Folley and French (1949) conducted

similar studies and observed considerably higher values,
due probably to their correction for milk content.

Just

prior to parturition the QOg had its lowest value of 1.5
but increased on the day following parturition (QOg = 4.0)
and reached its peak at mid-lactation (QOg = 6.3).

An in

vitro effect of prolactin was first shown by Folley (1952),
who observed that prolactin was capable of increasing net
respiratory activity only in lactating rat mammary slices
and not in mammary slices from pregnant rats.

Experiment

VI shows that prolactin can increase the oxygen consumption
of lactating rat slices in vitro.

It would have been

66

worthwhile to determine the in vitro effects of prolactin '
on oxygen consumption in mammary tissue from pregnant rats.

SUMMARY
1*

In Experiment I prolactin was incubated in vitro

for 1 hour at 38° C. with the following tissues:

liver,

kidney, brain and muscle of rats; mammary gland from lactat­
ing and nonlactating guinea pigs and rabbits; pigeon crop
glands; corpora lutea and the remaining ovarian tissue from
pseudopregnant rabbits.

After incubation the prolactin was

assayed in pigeons and directly compared with an equivalent
amount of prolactin which had been incubated in a tissuefree solution.
On a fresh tissue basis, mammary gland, pigeon crop
gland and the two ovarian tissues showed the greatest capacity
to inactivate prolactin.

Lactating mammary slices appeared

to be more effective in this respect than nonlactating mammary
slices.

Liver and kidney inactivated only about half as much

prolactin as the preceding tissues, while muscle and brain had
no effect on the prolactin.

Liver and kidney homogenates

were more effective in removing prolactin than slices of
these tissues.

When any of the tissues were boiled for 10

minutes prior to incubation with prolactin, they lost their
ability to inactivate the hormone.
2.

In Experiment II the inguinal nummary glands were

removed from 17 rats on the 12-15th days of gestation and
from 19 rats on the 4th day postpartum.

The glands were

68

homogenized and 100-mg. samples were incubated with 0,2 mg.
of Squibb prolactin (20-25 I.U./mg. ) at 38 ♦ 0.1° 0. for 1
♦

hour.

For controls, the same amount of prolactin was simi­

larly incubated without mammary tissue or with boiled
mammary tissue.

After incubation, the tissues were removed

and assayed in White Carneau pigeons for prolactin activity.
The mammary homogenate from the lactating rats inacti­
vated 65.6 per cent of the prolactin compared with 19.5 per
cent for the tissue from the pregnant rats.

When corrected

for milk content, by lactose determinations, the former
tissue showed 8 times as much capacity to remove prolactin
as the latter.

These data suggest that the mammary glands

of pregnant rats cannot utilize prolactin to any marked
extent, and that this contributes to the absence of copious
lactation during gestation.
3.

In Experiment III a total of 63 albino rabbits of

both sexes, intact and castrate, were injected with 960
I.U. of estrone and 1 I.U. of progesterone daily for 25
days in order to induce optimal mammary development.

From

the 26th to 35th days, all except three rabbits were in­
jected daily with 2 mg. of prolactin (20—25 I«U. per mg.),
with or without one or both of the steroid hormones.

At

the end of the 35th day, the rabbits were killed and the
mammary glands were exposed and rated for intensity of lac­
tation.

The extent of mammary growth induced by these treat­

ments was determined by injecting radioactive phosphorus
(P 33) 4 hours prior to sacrifice of the rabbits, and then
making r a d i o autographs of the mammary glands. *

69
The rabbits given prolactin and both steroids during
the last 10 days of the 35 day experimental period had prac­
tically no milk in their mammary glands (average rating of
less than l)> those injected with prolactin alone had mammary
glands filled with milk (average rating of 3 to 4); those
given prolactin and progesterone showed no inhibition in
lactational response (average rating of 3 to 4); and the
rabbits injected with prolactin and estrone showed only a
slight decrease in milk secretion (average rating of 2 to
3).

The radioautographs indicated the presence of intensive

mammary growth in the rabbits treated with both steroids
throughout the entire 35 day period but not in the rabbits
given both steroids only for the first 25 days.

It is con­

cluded that in the doses employed, estrone and progesterone
together can effectively inhibit the milk-secreting action
of prolactin on the mammary gland of the rabbit, whereas
progesterone alone is ineffective and estrone alone is only
slightly effective in this respect.
4.

In Experiment IV 36 young rabbits were ovariecto­

mized, and after 10 to 14 days were injected daily with
0.96 mg. of estrone and 1 mg. of progesterone for 25 days to
induce optimal mammary development.

From the 26th to 35th

days, 3 groups of 6 rabbits each were continued on the same
doses of steroid hormones together with 2, 4 and 8 mg. of
prolactin daily, respectively; 3 other groups of 6 each
were injected with 2 mg. of prolactin daily together with 0,
1/4 or 1/2 of the 2 steroid hormones used previously.

On

70

the 36th day the rabbits were killed and their mammary
glands were exposed and rated visually for lactational
response.
In the first 3 groups lactation was almost completely
inhibited when only 2 mg. of prolactin was injected daily in
the presence of the 2 steroid hormones, while 4 or 8 mg.
daily elicited good lactational responses.

In the second 3

groups, the greatest amount of milk was secreted when 2 mg.
of prolactin was given daily without the steroid hormones,
while the same dose of prolactin in the presence of either
1/4 or l/2 of the steroid hormones produced a smaller flow
of milk.
It is concluded that the antagonism between mammary
growth and lactation is relative, depending on the balance
between the levels of prolactin and the 2 ovarian hormones
in the body.

When the mammary growth stimulus exerted by the

2 ovarian hormones is greater than the lactational stimulus
of prolactin, milk flow will be inhibited, and vice versa.
The application of these findings to explain the absence of
lactation during a non-lactational pregnancy or the simul­
taneous coexistence of lactation and pregnancy is discussed.
5.

In Experiment V, extracts of mammary tissue taken

from parturient rats and rabbits were prepared and injected
subcutaneously into mature and lactating rats.

Thirty mature

rats were divided into three groups and were injected daily
for 10 days as follows*-

group 1 received a normal saline

71
solution; group 3 received 1.5 ml. rat mammary extract daily;

$ received

group

1.0 ml. rabbit mammary extract daily.

At

the end of the experimental period the rats were killed and
their pituitaries were assayed for prolactin content.

The

rat extract increased pituitary prolactin level by 41.8 per
cent, while the rabbit extract decreased it by 2 7.7 per cent.
Sixteen additional rats on the 4th day of lactation
were isolated for 4 hours from their litters and divided
into two groups of 9 and 6, respectively.

Group 1 received

1 ml. of .normal saline, while group 2 received 1.5 ml. rat
mammary extract in a single injection.

At the end of 24 hours

they were killed and their pituitaries were assayed for pro­
lactin content.

The rat mammary extract elicited a 16.9 per

cent increase in prolactin content.
On the basis of these limited data it appears that
mammary extracts from parturient rats and rabbits do not
influence'the pituitary prolactin content when injected into
mature and lactating female rats.

Further work is necessary

before a definite conclusion can be drawn regarding the
effects of mammary tissue on pituitary prolactin secretion.
6.

In Experiment VI, the jln vitro effects of prolactin

on the Q02 of mammary slices from lactating rats was deter­
mined.

Eighteen rats on the 4th, 5th, 8th, 9th, 10th, and

12th day of lactation were decapitated and their inguinal
mammary glands were removed.

was determined on slices in

Warburg flasks containing acetate-glucose-phosphate buffer
solution in the absence or presence of prolactin.

Prolactin

72
was found to increase oxygen consumption, particularly in
the latter stages of lactation.

This suggests that the

mammary glands from parturient rats are susceptible to pro­
lactin stimulation.
7.

Lactation is thought to be held in abeyance during

pregnancy by two mechanisms:

(l) the growth stimulating

levels of estrogen and progesterone on the mammary gland,
render it refractory to prolactin stimulation;

(2 ) the

relatively low level of prolactin secretion is insufficient
to overcome the inhibitory effects of the 2 ovarian hormones
on the mammary gland.

At parturition, the high levels of

the two ovarian hormones are greatly reduced, permitting the
increased amounts of prolactin from the pituitary to ini­
tiate lactation.

Lactation can continue during a subsequent

pregnancy because (a) prolactin secretion is maintained at
a high level by the milking act and (b) estrogen and pro­
gesterone are not secreted in sufficient quantities, parti­
cularly during the first half of pregnancy, to overcome the
action of prolactin on the mammary gland.

Other hormones

may also influence the initiation and maintenance of lacta­
tion, but they are believed to play a secondary role to the
factors mentioned above.

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