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INITIATION OF LACTATION IN RATS

BY NONSPECIFIC STRESSES

BY

CHARLFS SAMUEL NICOLL

AN ABSTRACT

Submitted to the college of Science and Arts,

Michigan State University of Agriculture and

Applied Science, in partial fulfillment of
the requirements for the degree of

MASTER OF SCIENCE

Department of Physiology'& Pharmacology

1960

 

CHARLES SMEUELEL NICOLL.
1

ABSTRACT

.L.

3.

Release of ACTH and luteotropin or prolactin from the anterior pit-
uitary may be induced by numerous factors such as stresses, adrenalectomy,
suckling, thyroxin, acetylcholine, serotonin, chlorpromazine, morphine,
epinephrine and reserpine. Since several of these factors are capable

of initiating lactation in rats and other Species, it was of interest

to determine whether several nonspecific stresses could promote release

of sufficient prolactin and ACTH to initiate lactation in the rat.

Sixty mature virgin female rats of the Carworth strain were divided

into 6 groups of 10 each and injected subcutaneously for 10 days with 10
‘pgm. estradiol in 0.1 cc corn oil. For the subsequent 5 days the groups
were treated as follows: 1) controls, 0.1 cc saline injected subcutaneously
once daily; 2) severe cold (00 C) 24 hours/day; 3) intense light and heat
(35°) for 12 hours/day; 4) restraint, 12 hours/day; 5) starvation, no

food or water for 5 days; 6) subcutaneous injection of 0.1 cc or 0.2 cc

of 10% formaldehyde once daily. On the 6th day the rats were killed and

the mammary tissue was removed from each rat for histological examination.

The adrenals, ovaries, uterus and thymus of each rat were weighed.

The mammary glands of the control rats regressed from a lobulo-alveolar
system to a bare duct system during the period of saline injections. In
the stressed groups lactation was initiated in 8 of the cold exposed, 7

of the light and heat treated, 9 of the restrained, 3 of the starved and 5
of the formalin injected rats. Thymic weight was significantly reduced in
all of the stressed groups and the adrenals were significantly enlarged in
the groups exposed to cold, starvation and restraint. This indicates

adrenal cortical stimulation in response to the stresses. Ovarian and

CHARLES SAMUEL NICOLL

2

ABSTRACT

uterine weights were significantly reduced in all stressed groups

with the exception of those exposed to cold.

It is ctncluded that certain nonspecific stresses can induce the
release of prolactin and ACTH from the anterior pituitary in amounts
sufficient to initiate lactation in the estrogen-primed rat. These
observations suggest that the stress of parturition may be involved

in the initiation of lactation which normally accompanies delivery.

The ability of numerous factors to promote release of prolactin
and ACTH from the anterior pituitary suggests that a canmon mechanism
may be involved in regulation of the secretion of both hormones. Such
a mechanism may involve a common neural regulatory center or centers

which are functionally associated.

INITIATION OF LACTATION IN RATS

BY NONSPECIFIC STRESSES

BY

CHARLES SAMUEL NICOLL

A THESIS

Submitted to the college of Science and Arts,

Michigan State University of Agriculture and

Applied Science, in partial fulfillment of
the requirements for the degree of

MASTER OF SCIENCE
Department of Physiology & Pharmacology
1960

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Note

The text of this thesis is a verbatim copy
of a manuscript which was submitted for
publication to the American Journal of
Physiology. Notification of acceptance

of the manuscript was received on January

3, 1960.

II

III

TABLE OF CONTENTS

INTRODUCTI‘DDJ OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

IMHODS 0.0......00.0.0000...O0.0...OOOOOOOOOOOOOOOIOOO

RESULTS .00...O0..OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOC

DISCUSSIOb‘ OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

PLEFERENCES Q...0.0...0.0.0.0...OOOOOOOOOOOOOOOOOOOOOOOO

APPENDIX 00.0.0000...0.000000...OOOOOOOOOCOOOO0.0.0....

Page

10

15

Table

1.

Figure

l.

3.

5.

Table

1A.

LIST OF TABLES

Effect of stresses on mammary glands
amihflyamimgmimflgms..u.n.n.n.n.n.n.u.n.

LIST OF FIGURES

Photomicrograph of control mammary tissue
from a rat injected with saline for 5 days
after 10 days of estradiol treatment ....................

Photomicrograph of mammary tissue from a
rat exposed to severe cold stress for 5
days after 10 days of estradiol treatment ...............

Photomicrograph of mammary tissue from a
rat exposed to intense heat and light stress
for 5 days after 10 days of estradiol treatment .........

Photomicrograph of mammary tissue from a rat
exposed to restraint stress for 5 days after
10 days of estradiol treatment ..........................

Photomicrograph of mammary tissue from a rat
exposed to starvation stress after 10 days of
eStradiOl treatment OOOOOOOOOOOOOQOOOOOOOOODOOOOOOOOOOOOO

Photomicrograph of mammary tissue from a rat

injected with formaldehyde after 10 days of
eStradiOl treatment OOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO.

APPENDIX

Effects of prolactin and ACTH on mammary glands
and body and organ weights ..............................

Page

Page

Page

17

List of Figures

Figure ‘Page
1A. Photomicrograph of mammary tissue from a rat treated
with 2 I. U. of ACTH and 2 mg. of prolactin for 5 days

following 10 days of estradiol injection.............. 18

2A. Photomicrograph of mammary tissue from a rat treated
with h mg. of prolactin per day for 5 days following

10 days Of eStradiOl injection..............o.......o. 18

OOO‘OI-Q-coooo

...U....'.O‘O...l9¢

Acknowledgments

The author wishes to express his sincere gratitude
to Dr. J. Meites, Professor, Department of Physiology and
Pharmacology, for his generous assistance. advice, stimilus
and constructive criticism throughout the course of this
investigation and during the preparation of this manuscript.
He also wishes to express his appreciation to Dr. B. V. Alfredson,
head of the Department of physiology and Pharmacology, for
providing facilities for conducting experiments. Special thanks
are due toer. P..K. Talwalker for assistance during the course
of the experiments and to Ems. L. F. Koci and Fun A. G» Stelma
for typing.

Many thanks are also due to Dr. E. P. Reineke, Dr. L. F.
Wolterink, Dr. R. F. Johnson. Dr. C. F. Cairy, Dr. J. E.
Nellor and Dr. W. D. Collings, all of the Department of PhysiOIOgy
and Pharmacology, And Dr. C. A. Hoppert, Department of Chemistry,
for instruction during the course of the author’s studies. The
author also wishes to thank MT. Merlyn Swab for assistance in
caring for experimental animals.

I am also indebted to the Michigan State University
Agriculture Experiment Station and the National Institutes
of Health for providing financial support to Dr. J. Nbites,

which enabled the investigation to be conducted.

INTRODUCTION

Release of ACTH from the anterior pituitary of the rat may be
induced by numerous factors such as various stresses (1), adrenalectomy
(2), the suckling stimulus (3), thyroxine administration (4), and in-
jections of numerous drugs including acetylcholine (5), serotonin (6),
chlorpromazine (7), morphine (8), epinephrine and reserpine (9). All
these factors have also been reported to produce physiological manifesta-
tions in the rat which are interpreted as indicative of increased secre-
tion of luteotropin or prolactin from the anterior pituitary. Thus
adrenalectomy (10), stresses (ll), epinephrine (ll), thyroxine (12),
suckling (13), reserpine and chlorpromazine (14) induced pseudopregnancy
in the rat. Lactation may be initiated in rats by the suckling stimu-
lus (13), and injections of morphine (15), epinephrine, acetylcholine,
serotonin and reserpine (16, 17) and numerous other agents which have
previously been reported to promote ACTH secretion (unpublished). Since
several of the factors which apparently elicit release of both ACTH and
prolactin are capable of initiating lactation in rats, it was of interest
to determine whether several nonspecific stresses could promote release

of ACTH and prolactin in amounts adequate to initiate lactation in rats.

METHODS

Sixty virgin female rats (Carworth) weighing 200 - 250 gms were
divided into 6 groups of 10 each and injected subcutaneously once daily
for 10 days with lO‘pgm estradiol in 0.1 cc corn oil. The estradiol
induces a variable degree of mammary lobulo—alveolar development, in
the rat, and is necessary to render the mammary tissue responsive to
hormones which stimulate lactation. For the following 5 days the rats
were treated as follows: 1) controls, 0.1 cc physiological saline in-
jected subcutaneously once daily; 2) severe cold (00 C) 24 hrs/day;

3) intense light and heat (35° C) produced by placing two 150 watt re-
flector floodlights over the cage containing the rats, for 12 hrs/day;
A) restraint produced by wrapping the tails or hind limbs of the rat
with several turns of masking tape, and then taping the tails or hind
limbs of 5 of the animals together for 12 hours per day. This pro-
cedure greatly hindered the movements of each animal and resulted in
considerable fighting among the rats. Preliminary trials with simple
restraint, produced by securing the forelimbs of the rats to their
thorax by several turns of masking tape, showed that this was not a
severe enough stress to initiate lactation: therefore, the more se-
vere method was adopted; 5) starvation, with no food or water for 5
days; 6) subcutaneous injection of 0.1 or 0.2 cc 10% neutral formalde-
hyde to 5 rats each. On the 16th day of the experiment the rats were
killed with nembutal and the right inguinal mammary gland was removed

and prepared for histological examination by standard techniques (17).

The ovaries, uterus, adrenals and thymus were also excised from all ani-

mals and weighed on a Roller - Smith balance.

RESULTS

The experimental data are summarized in Table I. When saline was
injected for 5 days following estrogen treatment (group 1), the mammary
glands regressed from a lobulo-alveolar to an almost bare duct system
in all cases. All the groups subjected to nonspecific stresses after
estrogen-priming showed maintenance of the mammary lobulo-alveolar

structure and lactation in varying degrees (Fig. 1 — 6).

The control rats (group 1) and those exposed to cold (group 2) or
light and heat (group 3) showed no change in body weight during the ex-
perimental period. The starved rats (group 5) lost an average of 58
grams each and those subjected to restraint (group 4) lost an average
of 10 grams each. The rats given 0.1 cc formaldehyde (group 6A) gained
an average of 30 grams but showed no weight change when given 0.2 cc
(group 6B). Formaldehyde produced large edematous pockets at the site
of injection. The 0.2 cc dose of formaldehyde produced a harsher stress
than the 0.1 cc dose as indicated by adrenal and thymus weights. The
adrenals were significantly enlarged in the groups exposed to cold,
starvation and restraint while the rats treated with 0.1 cc formalin
showed a decrease in adrenal weight per 100 gms body weight. This is
probably due to the relatively large body weight gain in this group.
All the stressed rats showed reduced thymus weights which is indicative
of increased adrenal cortical secretion. Ovarian and uterine weights

were significantly decreased in all stressed rats with the exception of

those exposed to cold; this can be considered indicative of decreased

gonadotropic secretion, previously reported to occur under stress (18).

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Figure 1. Photomicrograph of control mammary tissue from a
rat injected with saline for 5 days after 10 days
of estradiol treatment.

X 130.

Figure 2. Photomicrograph of mammary tissue from a rat exposed
to severe cold stress for 5 days after 10 days of estrodiol
treatment.

X 130.

 

 

Figure 1

 

Figure 3. Photomicrograph of rat mammary tissue frem a rat

exposed to heat and light stress for 5 days after

10 days of estradiol treatment.

Figure A. Photomicrograph of rat mammary tissue from a rat
exposed to restraint stress for 5 days after 10

days of estradiol treatment.

 

 

 

 

Figure 5. Photomicrograph of mammary tissue from a rat exposed
to starvation stress for 5 days after 10 days of estradiol
treatment.

X 130.

Figure 6. Photomicrograph of mammary tissue from a rat injected
with formaldehyde for 5 days after 10 days of estradiol
treatment.

X 130.

 

 

10

DISCUSSION

It is evident that certain nonspecific stresses can induce the
release of prolactin and ACTH from the anterior pituitary in amounts
sufficient to initiate secretory activity in the mammary glands of
the estrogen—primed rat. This is of interest when one considers pre-
vious reports that stresses inhibit nonessential functions such as
growth, reproduction and lactation (19). Administration of large
doses of ACTH or glucocorticoids for 10 days has been observed to
initiate slight lactation in untreated virgin rats (20). We have
recently observed, however, that daily injection of 2 I.U. or 4
I.U. of ACTH for 5 days following estrogen priming failed to induce
lactation in rats despite obvious stimulation of the adrenal cortex.
Injection of 2 mg. or 4 mg. of prolactin (2O I.U. per mg) per day
after estrogen priming also failed to initiate secretion in the mam-
mary glands of these rats. However, daily injections of 2 I.U. ACTH
and 2 mg prolactin together was effective in inducing secretion in
these rats (unpublished)*. We believe these observations indicate
that the induction of lactation in rats with nonspecific stresses is
not merely the result of enhanced adrenal cortical activity, but also

of augmented secretion of prolactin from the anterior pituitary.
Selye (21) has previously reported that lactation was initiated

(* See Appendix)

11

in spayed rats when estradiol was administered with cortisol or large
doses of ACTH for 8 days, but kaolin or formaldehyde injections in
conjunction with estradiol failed to induce mammary secretion. It is
difficult to rationalize Selye's observation with those of the present
study since the experimental conditions varied considerably and the

type and severity of the stresses employed are not strictly comparable.
Possibly the use of intact rats and sequence of treatment in the pres-
ent study accounted for the differences. Initiation of lactation by
stresses in the estrogen—primed rat may be related to clinical reports
of induction of lactation in women after thoracoplasty (22), simple
appendectomy (23), severe burns, starvation and other harsh trauma (24).
Ovariectomy has also been reported to induce lactation in pseudopregnant
rats (25) and in women (26) and has been attributed to the hormonal with-
drawal phenomena. However, surgical trauma during ovarectomy may also

be partially responsible for initiating lactation in these cases.

Recent theories concerned with the initiation of lactation at
parturition have emphasized the changing levels of circulating ovarian
hormones at term (27). High blood levels of estrogen and progesterone
during gestation are believed to render the mammary gland relatively
refractory to prolactin stimulation, and the predominance of proges-
terone over estrogen is believed to prevent the latter from increasing
prolactin secretion. At the end of pregnancy, the inhibition of the
two hormones on the mammary glands is decreased, and estrogen is be-
lieved to become sufficiently predominant to increase prolactin se-
cretion and initiate lactation. The ability of nonspecific stresses

to initiate lactation suggests that the stress of parturition may also

12

have a role in promoting the copious milk flow which normally accompanies

delivery.

In view of the apparent ability of nonspecific stresses and numer-
ous other factors to promote secretion of prolactin and ACTH, it is pos—
sible that a common mechanism can influence the secretion of both hor-
mones. There is evidence that the CNS normally inhibits prolactin se-
cretion, as indicated by persistent secretion of prolactin from rat
pituitary autografts (28). There is also evidence that the midbrain
of the rat contains inhibitory and stimulatory centers for ACTH secre-
tion (29). If the neural inhibition of prolactin and ACTH involves
the same CNS center, or centers which are functionally associated, the
numerous factors Which promote the secretion of these hormones may op—
erate by suppressing the inhibitory center(s). In accordance with
this view, the autotransplanted pituitary would continue to elaborate
prolactin since the neural inhibition would be removed, and the secre-
tion of ACTH would diminish because the CNS stimulatory center would
no longer be effective. Such a concept is compatible with the re-
sults of the present study and with other circumstances were prolactin

and ACTH are released concomitantly.

10.

11.

12.
13.
14.
l5.

l6.

l7.

13

REFERENCES

Selye, H. and G. Heuser. 5th Annual Rgport on Stress. Montreal,
Canada. Acta Inc. 1955/56.

 

Sydnor, K. L. and G. Sayers. Endocrinology 55:621, 1954-
Gregoire, C. J. Endocrinology 5:68, 1947.
Eartly, H. and C. P. Leblond. Endocrinology 54:249, 1954.

Casentini, S., A. DePoli, S. Hukovic and L. Martini.
Endocrinology 64:483, 1959.

Moussatche, H. and A. N. Pereira. 'Agta Physiol. Latino-Am.
7:70, 1957. (Cited by I. H. Page. Physiological Reviews
38:327, 1958)

Halzbauer, M. and M. Vogt. Brit. J. Pharmacol. 9:402, 1954.

Szerb, J. C. Can. J. Med. Sci. 31:8, 1953.

 

Kitay, J. I., D. A. Holub and J. W. Jailer. Endocrinology.
653548, 1959-

Swingle, W. W., E. J. Fedor, G. Barlow, Jr., E. J. Collins and
J. Purlmutt Am. J. Physiol. 167:593, 1951.

Swingle, W. W., P. Seay, J. Purlmutt, E. J. Collins, G. Barlow,
Jr., and E. J. Fedor. Am. J. Physiol. 167:586, 1951.

Selye, H. and T. McKeown. Surgi Gyn. and Obst. 59:886, 1934.

 

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

Meites, J., C. S. Nicoll and P. K. Talwalker. Proc. Soc. Exp.
Biol. and Med. 102:127, 1959.

 

Meites, J. Proc. Soc. Exp, Biol. and Med. 100:750, 1959.

Meites, J., C. S. Nicoll and P. K. Talwalker. Proc. Soc. Exp.
Biol. and Med. 101:563, 1959.

 

'5 ' '—-——L“

18.
19.

20.

21.

22.

23.

25.

26.

27.

28.

29.

14

Selye, n. Endocrinology. 25:615, 1939.

 

Selye, H. Endocrinology. 49:197, 1951.

Johnson, R. M. and J. Meites. Proc. Soc. Exp, Biol. and Med.
89:455, 1955.

 

Selye, H. Rev. Can. de Biol. 13:377, 1954.

Salkin, D., W. V. Hopement and B. W. Davis. J. Thorac. Surg.
18:580, 1949.

 

Giusti, A. Boll. Soc. Med. Chir. Medina 1947., (Cited by H.
Selye lst Annual Report on Stress, Montreal, Canada. Acta
Inc. 1951, p. 199 )

 

Bartolomei, G. Riv. Ostet. e Ginec. :35, 1953. (Cited by H.
Selye. 4th Annual Report on Stress. Montreal, Canada.
Acta Inc. 1954, p. 407.

 

Meites, J. and C. W. Turner. Mo. Arg; Exp. St. Bull. 415:39,
1948.

 

Selye, H. lst. Ann. Report. on Stress. Montreal, Canada. Acta
Inc. 1951, p 199.

Meites, J. Chapt. 16 in Reproduction in Domestic Animals
(H. H. Cole and P. T. Cupps, Eds.) New York. Academic Press.
1959, p. 554.

Everett, J. W. Endggpipplogy. 58:786, 1956.

Slusher, M. A. and V. Critchlow. Proc. Soc. Exp. Bipl. apd
Med. 101:497, 1959.

 

 

15

APPENDIX

In order to test the hypothesis that stress induced lactation in
the estrogen—primed rat is due to augmented secretion of prolactin and
ACTH from the anterior pituitary, and not merely the result of enhanced F
adrenal cortical activity, ACTH and prolactin were administered singu-

larly and in combination to estrogen primed rats.

 

Twenty—five mature virgin female rats were divided into 5 groups \

of 5 each and injected subcutaneously for 10 days with 10 pgm of es-

tradiol in 0.1 cc of corn oil. For the subsequent 5 days the respec—

tive groups were injected twice daily with the following; 1) 1 mg of

prolactin; 2) 2 mg of prolactin; 3) 1 I.U. of ACTH; 4) 2 I.U. of ACTH;

5) 1 mg of prolactin and 1 I.U. of ACTH. All injections were given

subcutaneously. 0n the 16th day of the experiment the right inguinal

mammary glands were removed from each rat for histological examination

and the adrenals, thymus, ovaries and uterus were excised from groups

2 - 5 and weighed on a Roller-Smith balance. The uteri from group 3

were not weighed.

As may be seen from the accompanying table (Table 1A), neither
prolactin nor ACTH were effective in initiating lactation in the es—
trogen primed rat when given alone. However, when ACTH and prolactin
were administered together (Fig. 1A) lactation was initiated in 4 of
the 5 rats, thus providing support to the hypothesis that stress in-

duced lactation in the estrogen-primed rat is the result of enhanced

l6

prolactin and ACTH secretion and not to increased ACTH secretion
alone. Although neither dose level of prolactin was effective in in-
itiating lactation in the estrogen—primed rat, the 4 mg per day level

retarded mammary involution in all of the 5 rats treated (Fig. 2A).

Both dose levels of ACTH effectively stimulated the adrenal cor—
tex of the rats as judged by the thymolytic effects which are compar—
able to those observed in the stressed rats. The 2 I.U. per day level
of ACTH resulted in a 40.5% reduction and the 4 I.U. per day level re—
sulted in a 48.1% reduction in thymic weight in comparison with the
control weight. The stressed rats exhibited a range of thymic weight
reduction from 19.6% for the starved rats to 54.9% for the restrained

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Figure 1A. Photomicrograph of mammary tissue from a rat treated
with 2 I. U. of ACTH and 2mg. of prolactin for 5 days

following 10 days of estradiol injection. X 130.

Figure 2A. Photomicrograph of mammary tissue from a rat treated
with 4mg. of prolactin per day for 5 days following

10 days of estradiol injection. X 250.

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