A STUDY or DRUGS THAT INHIBIT
PROLACTIN SECRETION

Thesis for the Degree of M. S.
MICHIGAN STATE UNIVERSITY
MARIE CATHERINE GELATO
197 1

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ABSTRACT
A STUDY OF DRUGS THAT INHIBIT
PROLACTIN SECRETION
BY

Marie Catherine Gelato

l. The effects of pentobarbital (PB) on the pituitary
and on hypothalamic PIF activity were studied in female
rats. An injection of PB completely depleted the hypo-
thalamus of PIF activity by the end of 10 minutes, and 2
hours later PIF activity was still lower than in control
rats injected with saline. Addition of PB to a pituitary
incubation system resulted in inhibition of prolactin re-
lease, indicating that PB can directly inhibit prolactin
release by the pituitary. The initial brief rise in serum
prolactin observed after injecting PB is believed to be
due to a rapid decrease in hypothalamic PIF activity,
whereas the subsequent prolonged’fall in prolactin is
believed to be due to a direct inhibitory action on the
pituitary.

The effects of another barbituric acid derivative,
thalidomide, was determined on serum and pituitary prolac-

tin levels on the afternoon of proestrus. All three doses

Marie Catherine Gelato

used,!5,10, and 25 mg/lOO gm body weight, completely blocked
the proestrous afternoon surge. A dose—response effect of
thalidomide was indicated, since the highest dose of
thalidomide used produced the greatest blockage of the pro—
estrous prolactin peak, whereas serum prolactin levels were
progressively increased when lower doses of thalidomide were
used. Pituitary prolactin concentrations were increased
in all thalidomide—treated animals as compared to controls.
Thalidomide, like PB, decreased serum prolactin levels and
blocked the proestrous afternoon surge. It is possible that
thalidomide exerts its inhibitory effect on prolactin re-
lease by directly blocking the pituitary.

2. A single intraperitoneal injection of iproniazid
on the afternoon of proestrus, or cocaine on the morning of
proestrus, in female rats caused a significant reduction in
serum prolactin levels. Iproniazid completely blocked the
afternoon proestrus rise of prolactin whereas cocaine
caused a reduction in the pre-peak values of prolactin on
the morning of proestrus. Several doses of cocaine were
tested, and in some cases high doses of cocaine first in-
hibited and then stimulated prolactin release. All doses of
cocaine used had no effect on pituitary prolactin concen-
tration as compared to controls. The inhibitory effects of
iproniazid and c0caine on prolactin release may be related
to their demonstrated ability to increase catecholamine

levels in the hypothalamus.

Marie Catherine Gelato

3. Daily injections of ergocornine, 400 ng and 800
ug per rat, a dose known to suppress both prolactin and LH
levels, were given to female rats for one estrous cycle.
Sixteen of the 32 rats also were given a single injection
of 1 mg of prolactin on the afternoon of proestrus. ~As a
result of the ergocornine treatment the size of the ovaries
and the number of corpora lutea were significantly increased
as compared to controls. The animals receiving both ergo—
cornine and prolactin showed no change in ovarian weight or
number of corpora lutea when compared to controls.

Similar experiments using iproniazid were carried out
for one and three estrous cycles. The animals given iproni-
azid in both studies showed an increased number of corpora
lutea and increased ovarian weight. Iproniazid-prolactin
treated animals showed no change in ovarian size or number
of corpora lutea as compared to controls. These experiments
add further proof for the role of prolactin as a luteolytic

agent during the estrous cycle.

A STUDY OF DRUGS THAT INHIBIT

PROLACTIN SECRETION

BY

Marie Catherine Gelato

A THESIS

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

MASTER OF SCIENCE

Department of Physiology

1971

Dedicated
to my

Mother and Father

ii

ACKNOWLEDGEMENTS

The author wishes to express sincere gratitude to
Dr. Joseph Meites for his guidance and encouragement during
the course of this study. An expression of thanks is also
extended to the members of the guidance committee, Dr. H.
Hafs, Dr. R. Bernard and Dr. T. Jenkins for their advice
and assistance in the preparation of this manuscript.

The author is indebted to Dr. T. Brody, Professor of

Pharmacology, who kindly supplied the cocaine used in this

study.

 

iii

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW. . . . . . . . . . . . . . . . . . 3
I. Functions of Prolactin. . . . . . . . . . . 3
II. Hypothalamic Inhibition . . . . . . . . . . 9
III. Catecholamines. . . . . . . . . . . . . . . 12
IV. Iproniazid. . . . . . . . . . . . . . . . . 14
V. Cocaine . . . . . . . . . . . . . . . . . . 16
VI. Barbiturates. . . . . . . . . . . . . . . . 17
VII. Ergot Drugs . . . . . . . . . . . . . . . . 19
VIII. Effects of Hormones . . . . . . . . . . . . 20
MATERIALS AND METHODS. . . . . . . . . . . . . . . . 23
I. Animals 0 O O O O O O O O O O O O O O O O O 23
II. Treatment of Pituitary and Blood Samples. . 23
III. Radioimmunoassay. . . . . . . . . . . . . . 24
IV. Histological Treatment. . . . . . . . . . . 24
V. Statistical Treatment . . . . . . . . . . . 25
EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . 26
I. Effects of Barbiturates on Prolactin
Secretion . . . . . . . . . . . . . . . . . 26
A. Effect of Injecting Na Pentobarbital
(PB) on Hypothalamic PIF Activity and
Effect of Na Pentobarbital on Pituitary
Prolactin Release In Vitro in the Rat . 26
1. Objective . . . . . . . . . . . . . 26
2. Procedure . . . . . . . . . . . . . 26
3. Results . . . . . . . . . . . . . . 27
B. Effects of Thalidomide on Serum and
Pituitary Prolactin Levels in the Rat . 30
1. Objective . . . . . . . . . . . . . 3O
2. Procedure . . . . . . . . . . . . . 3O
3. Results . . . . . . . . . . . . . . 30
C. Conclusions . . . . . . . . . . . . . . 32

iv

TABLE OF CONTENTS—-continued Page
II. Effects of Drugs that Alter Hypothalamic
Catecholamines on Prolactin Release . . . . 33

A. Effects of a Single Injection of
Iproniazid on Pituitary Prolactin

Release . . . . . . . . . . . . . . . . 33
1. Objective . . . . . . . . . . . . . 33
2. Procedure . . . . . . . . . . . . . 33
3. Results . . . . . . . . . . . . . . 34
B. Effects of Cocaine on Pituitary and
Serum Prolactin Levels. . . . . . . . . 34
1. Objective . . . . . . . . . . . . . 34
2. Procedure . . . . . . . . . . . . . 34
3. Results . . . . . . . . . . . . . . 36
C. Conclusions . . . . . . . . . . . . . . 38

III. Luteolytic Role of Prolactin During the

Estrous Cycle in Female Rats. . . . . . . . 39

A. Effects of Ergocornine (EC) Treatment . 39

1. Objective . . . . . . . . . . . . . 39

2. Procedure . . . . . . . . . . . . . 39

3. Results . . . . . . . . . . . . . . 40

B. Effects of Iproniazid Treatment . . . . 42

1. Objective . . . . . . . . . . . . . 42

2. Procedure . . . . . . . . . . . . . 42

3. Results . . . . . . . . . . . . . . 43

C. Conclusions . . . . . . . . . . . . . . 46
DISCUSSION . . . . . . . . . . . . . . . . . . . . . 49
LIST OF REFERENCES . . . . . . . . . . . . . . . . . 53

TABLE

LIST OF TABLES

Page
Effect of Injecting Pentobarbital (PB) on
Hypothalamic PIF Activity
Effect of PB on Pituitary Prolactin Release
ig_vitro . . . . . . . . . . . . . . . . . . . 29
Effects of Thalidomide on Serum and Pituitary
Prolactin Levels . . . . . . . . . . . . . . . 31
Effects of Iproniazid on the Proestrous Rise
Of PrOJ-actin O O O O O 0 O O O O O O O O O O O 35
Effects of Cocaine on Serum and Pituitary
Prolactin Levels . . . . . . . . . . . . . . . 37
Effects of Ergocornine Injections During One
Estrous Cycle on Weight of Endocrine Organs
and Average Number Of Corpora Lutea. . . . . . 41
Effects of Iproniazid Injections During One
Estrous Cycle on Ovaries . . . . . . . . . . . 44
Effects of Iproniazid Injections During Three
Estrous Cycles on Ovaries. . . . . . . . . . . 45
Effects of Iproniazid Injections During Three
Estrous Cycles on Endocrine Organs . . . . . . 47

vi

INTRODUCTION

Drugs have served as tools for studying the control
and functions of prolactin and other pituitary hormones.
Reviews by Meites (1962; 1963) and Coppola (1971) have
shown the importance of drug studies in elucidating prolac—
tin physiology. Drugs have been used to demonstrate the
relationship of the hypothalamus and its hypophysiotropic
hormones or factors to prolactin release and have led to a
better understanding of the role of prolactin in lactation,
mammary deveIOpment, mammary tumorigenesis, and ovarian
function.

Recently it was reported (Wuttke and Meites, 1970)
that Na pentobarbital initially stimulated and then in-
hibited prolactin release. Therefore, it was of interest
to determine the site of action of Na pentobarbital by
studying its effects on the hypothalamus and pituitary.
Since Na pentobarbital is a barbituric acid derivative, it
also was of interest to know if another drug in this class,
thalidomide, would have the same effect on prolactin release.

Brain catecholamines recently have been demonstrated
to influence the release of prolactin and other pituitary

hormones. An increase of catecholamine levels in the

hypothalamus is believed to inhibit prolactin release,
while a decrease of catecholamine levels in the hypothala-
mus is believed to stimulate prolactin release. Certain
drugs can be injected to alter catecholamine levels in the
hypothalamus. Iproniazid blocks the enzyme monoamine oxi-
dase and cocaine blocks the reuptake of catecholamines
by nerve endings. Through these mechanisms both drugs in-
crease catecholamine concentrations in the hypothalamus.
Because of these actions of iproniazid and cocaine on brain
catecholamine levels, it was of interest to study their
effects on prolactin release.

Prolactin levels in the serum rise on the afternoon
of proestrus together with LH and FSH, and remains high on
the morning of estrus during each cycle. Suppression of
this rise of prolactin by ergocornine resulted in a sig-
nificant enlargement of the ovaries due to the accumulation
of many old corpora lutea, whereas administration of prolac—
tin prevented these effects of ergocornine (Wuttke and
Meites, 1971). It was postulated from this work that the
rise of prolactin during the estrous cycle causes the luteo-
lysis of the old crop of corpora lutea from the previous
cycle. It was of interest therefore to determine the effects
of high doses of ergocornine known to suppress both prolac-
tin and LH on luteal function, and also to observe whether
another drug that depresses prolactin secretion, iproniazid,

could produce the same effects as ergocornine.

LITERATURE REVIEW

I. Functions of Prolactin

Prolactin is one of six hormones secreted by the anter-
ior pituitary. In mammals it has been closely associated
with growth hormone (GHf'and has growth promoting properties
in some species (Bern and Nicoll, 1968). Both prolactin and
growth hormone are synthesized and released by acidophils
within the anterior pituitary (Meites, 1966). Prolactin is
a large polypeptide, and only the structure of ovine pro-
lactin has been determined thus far (Li gt 31., 1969).

The role of prolactin in mammals has become defined
more clearly in the last decade. It has been well estab-
lished that prolactin plays a major part in normal and neo-
plastic mammary gland development, lactation, and luteal
function in the rat, mouse, ferret and hamster (Meites and
Nicoll, 1966). Since the literature on prolactin physi—
ology is vast, I will attempt to present some of the most
important and classical references. Other more detailed
reviews include those by Cowie and Folley (1955), Meites
(1962), Nicoll and Bern (1964), Meites and Nicoll (1966)

and Bern and Nicoll (1968).

The ovaries and the anterior pituitary are the two
most important organs in the control of growth and develop—
ment of the mammary glands. The classic experiments by
Lyons and co-workers (1958) used hypophysectomized, ovari-
ectomized, and adrenalectomized rats as a model, and showed
that injections of estrogen and GH produced duct growth
whereas injections of estrogen, progesterone, GH and pro-
lactin elicited lobulo-alveolar growth. These studies were
confirmed by the in_yit£g experiments of Elias (1957) and
Rivera (1964). Undeveloped mammary glands from mice and
quinea pigs were cultured for 5-7 days with combinations of
insulin, estrogen, progesterone, GH and prolactin, and these
mammary glands showed lobulo-alveolar growth.

However, other evidence indicates that anterior pitui-
tary hormones alone, prolactin and GH, can induce mammary
lobulo—alveolar growth equivalent to that seen in pregnancy
in the absence of ovarian hormones. Transplantation of a
pituitary mammotrOpic tumor into adreno-orchidectomized rats
of the inbred Fischer strain resulted in mammary lobulo-
alveolar growth equivalent to that seen during pregnancy
(Clifton and Furth, 1960). Related experiments by Talwalker
and Meites (1961) showed that prolactin and GH injections
into randomly bred Sprague—Dawley rats are enough to induce
lobulo-alveolar growth in the mammary glands. They used
rats which were adrenalectomized and ovariectomized or

adrenalectomized, ovariectomized and hypophysectomized and

gave injections of GH and prolactin or prolactin alone, and
observed considerable lobulo-alveolar growth by either
treatment. In sexually immature female rats hyp0physectom-
ized and implanted in the right inguinal region with an
anterior pituitary from a mature rat, mammary lobulo-alveolar
growth occurred locally only near the implanted area. The
transplanted pituitary has been shown to release both pro—
lactin and GH (Meites and Kragt, 1964).

It seems evident that prolactin and GH are the pre—
dominant factors in mammary gland development. It has been
demonstrated by Turner (1939), Lyons gt a1, (1958) and by
Meites and Hopkins (1961), that ovarian hormones have no
ability to stimulate mammary gland development in the ab-
sence of anterior pituitary hormones. It has been suggested
by Meites and Nicoll (1966) that the gonadal steroids stimu-
~1ate secretion of anterior pituitary hormones (prolactin and
GH), and synergize with the pituitary hormones to sensitize
the mammary tissue to these hormones.

In addition to its role in normal mammary deve10pment,
prolactin plays an important part in the development of
mammary tumors. Huggins (1965) reported that a single intra-
venous injection of 7,12-dimethy1benzanthracene (DMBA) into
female Sprague—Dawley rats, 50—60 days old, was able to
produce mammary tumors. This has become a useful model for
studying mammary cancer. With this preparation it is

possible to study the relationships between mammary tumors,

the hypothalamus, the anterior pituitary and the ovaries.
Ovariectomy, performed before carcinogen treatment, pre—
vents tumor induction (Huggins gt all, 1961; Dao, 1962;
Talwalker gt al.,1964; Welsch §§_§l,, 1968). However, it
lengthens mean latency period and reduces incidence of
tumors when performed after carcinogen treatment but before
tumor appearance (Huggins §E_al,, 1959). After mammary
tumors have developed, ovariectomy causes regression of
these tumors (Huggins §t_§l,, 1959; Sterental §t_§l,, 1964).
Hypophysectomy has a more drastic effect on mammary tumors
in the rat (Huggins §t_al., 1959; Kim 25 al., 1960; Dao,
1964; Sterental £3 31., 196472 Both ovariectomy and hypo—
physectomy have been shown to depress prolactin secretion
(Pearson £3 31., 1969; Chen gt_al., 1970). The current view
is that ovariectomy and hypOphysectomy depress or remove
prolactin levels, resulting in suppression of growth of
mammary tumors (Kim gt al., 1960; Meites, in press). It
also has been shown that injections of prolactin into rats
enhances growth of existing DMBA induced tumors and develop-
ment of new mammary tumors (Meites 2; 31., in press).
Initiation and maintenance of lactation is dependent
on adequate amounts of prolactin and adrenal corticosteroids.
Other hormones, including GH, thyroid stimulating hormone.
parathormone, and insulin, also may be necessary to promote
maximum milk production (Meites and Nicoll, 1966). In hypo-

physectomized guinea pigs, prolactin and adrenal cortical

hormones represented the minimal requirements for milk
secretion (Folley and Young, I941; Nelson §E_al,, 1943).
However, in intact rats (Talwalker gt $1., 1961) and mice
(Nandi and Bern, 1961) the corticosteroids rather than
prolactin were the limiting factor for initiating lactation
during pregnancy, since administration of adrenal corti-
coids alone was effective in initiating lactation whereas
prolactin alone was ineffective. In mammary tissue cultures,
insulin as well as prolactin and corticosteroids were found
to be necessary to initiate and maintain mammary secretion
(Rivera, 1964). A rise in prolactin and ACTH secretion has
been reported to occur at about the time of parturition
(Meites, 1959; Cowie and Folley, 1955) and both serve to
initiate lactation.

Prolactin also functions as a luteotropin but it may
act synergistically with LH. It maintains the corpus luteum
of the ovary. Astwood (1941) demonstrated in hypOphysectom—
ized rats, that administration of prolactin produced func-
tional corpora lutea. It has been shown that in rats the
luteotropic effect of prolactin is necessary, during the
first six days of pregnancy to maintain progesterone secre-
tion by the corpora lutea (Clemens gt 21,, 1969). It seems
that increased levels of prolactin tend to maintain func-
tional corpora lutea. EVerett (1956) showed that removal
of the anterior pituitary from its connection with the dien—

cephalon and placing it under the kidney capsule causes an

increased secretion of prolactin and prolongs luteotropic
activity in rats. Subsequent studies by NikitovitchAWiner
and Everett (1958) in which anterior pituitary grafts were
placed under the kidney and in the anterior chamber of the
eye, also demonstrated a prolonged luteotropic activity in
rats. Chlorpromazine and reserpine (Barraclough and Sawyer,
1959) or perphenazine (Ben—David, 1968) administration to
rats also prolongs luteotropic activity. .However, as men-
tioned by Meites and Nicoll (1966) prolactin can not be con-
sidered the only hormone involved in luteal function since
luteinizing hormone (LH) has been shown to be an important
regulator of luteal function in several species, including
the rat.

Prolactin has the ability to be luteolytic as well as
luteotropic in rats (Malven and Sawyer, 1966). .Hypophy-
sectomized rats were treated with LH and FSH in order to
induce ovulation and luteal formation. Prolactin injections
were given several days later and structural luteolysis
occurred whereas prolonged treatment with FSH and LH had no
effect on luteolysis. Further work by Malven (1969) demon-
strated both a luteotrOpic and luteolytic response to pro-
lactin in hypophysectomized rats. However, the function of
prolactin in a normal cycling rat was unknown until recently.
Radioimmunoassay of prolactin in the blood serum has shown
that it is highest on the afternoon of proestrus and remains

elevated in early estrus; it is low during most of pregnancy

(Amenomori gt 31., 1970; Gay £5 31., 1970). It has been
demonstrated very recently by Wuttke and Meites (1971) that
this rise of prolactin during the normal cycle serves to
induce luteolysis of the older crop of corpora lutea during
each cycle. The normal rise of prolactin on the afternoon
of proestrus and estrus was blocked by a drug, ergocornine.
A dose of ergocornine was used that is known to markedly
decrease prolactin levels but has relatively little effect
on LH levels and does not interfere with regular cycling

or ovulation (Wuttke gt 31., 1971). Luteolysis was blocked
in those animals treated with ergocornine. When exogenous
prolactin was administered on the afternoon of proestrus or
on the days of proestrus and estrus, luteolysis occurred as
determined by the number of corpora lutea and ovarian weight.
Related experiments by Billeter and Fluckiger (1971) are in
agreement with those presented above, although carried out
over a longer time period. Further evidence for the luteo-
lytic action of prolactin in the normal cycling rat will be

presented in this thesis.

II. .Hypothalamic Inhibition

 

The hypothalamus is intimately involved in regulation
of secretion of all the hormones from the anterior pituitary.
However, the regulation of prolactin secretion is unique in
that it is the only anterior pituitary hormone that is

chronically inhibited by the hypothalamus. The hypothalamus

10

produces a neurohumor, termed appropriately prolactin inhibib
ting factor (PIF), which traverses the hypothalamo-hypo-
physeal portal system and acts directly on the anterior
pituitary to inhibit prolactin synthesis and release
(Talwalker 2E.§l-c 1963).

Any disturbance in the connection of the hypothalamus
with the anterior pituitary can cause an increased secretion
of prolactin. Various experimental techniques have been
employed to accomplish the latter, such as pituitary stalk
section and administration of various CNS—depressant drugs
(Meites gt 11., 1963). Recently, it was reported that trans-
plantation of a pituitary beneath the kidney capsule of
hypophysectomized-ovariectomized rats or placement of elec-
trolytic lesions in the median eminence of ovariectomized
rats, resulted in increased serum prolactin levels (Chen
[gt al., 1970).

Culture of anterior pituitary tissue ig_yit£9_has demon-
strated that the anterior pituitary can synthesize and
release prolactin when removed from the hypothalamus and
other body influences (Meites, 1959). Other in_yit£g work
with incubated pituitaries and hypothalamic and cerebral
extracts, resulted in decreased release of prolactin only
when the pituitary tissue was incubated with hypothalamic
extract (Talwalker g; 31., 1963). Pasteels §t_gl, (1963)

did similar experiments with human pituitaries and observed

the same results. This work has been confirmed in vivo by

ll

Grosvenor gt a1. (1964) who injected hypothalamic extracts
into postpartum lactating rats and reported that they in—
hibited the usual prolactin release following suckling.

There are several stimuli which are known to increase
prolactin secretion and these have been employed to measure
PIF activity. Ratner and Meites (1964) demonstrated that
estradiol and suckling increased prolactin secretion by
depleting the hypothalamus of PIF activity. In a similar
series of experiments Sar and Meites (1967) showed that PIF
activity during proestrus and estrus in rats was less than
during the stage of diestrus. This corresponds to the
serum prolactin levels during these phases of the cycle
(Amenomori gt al,, 1970; Wuttke and Meites, 1970).

While the existence of PIF has been well documented in
mammals, its presence has not been demonstrated in other
species. Meites and Nicoll (1966) reported the presence of
a prolactin releasing factor (PRF) in 6 species of birds.
The chemical structures of these substances have not been
determined. However, this is a very active area of neuro-
endocrine research and it is very likely that within the
next few years the structures for most of the hypophysio—
tropic hormones of the hypothalamus will be determined.
Very recently the structures for thyrotrOpic releasing factor
(TRF or TRH) (Boler gt al,, 1969) and luteinizing hormone
releasing factor (LRF) (Matsuo gt al,, 1971) have been
reported; also that for melanocyte stimulating hormone

inhibiting factor (MIF) (Nair §t_§l., 1971).

12

III. Catecholamines

In the last few years much attention has been given
to the role of the catecholamines on gonadotropic secretion.
As mentioned earlier, the hypothalamus serves as a regula-
tor of the function of the anterior pituitary and possibly
the release of the hypothalamic hormones is mediated
through these catecholamines. There is much controversy as
to which of these biogenic amines, norepinephrine or dop-
amine, has the major role in release of pituitary hormones.
In an excellent review by Fuxe and Hokfelt (1969), much of
the current research on this question was presented.
Evidence is mounting in favor of dopamine as the major neuro—
transmitter substance. It was postulated by Coppola §E_gl.
(1966) that a sympathetic tOnus, originating in the hypo—
thalamus, normally acted to stimulate the release of FSH
and LH while restraining the secretion of prolactin. In the
absence of this tonus, FSH and LH secretion were suppressed
while prolactin release was stimulated. Evidence for this
postulate came from pharmacological studies in which certain
drugs which interferred with or enhanced catecholamine
activity were used, and gonadotropin secretion was monitored.

An early study by Barraclough and Sawyer (1959) demon—
strated that reserpine, a catecholamine depletor, and
Chlorpromazine, a potent adrenergic blocker, induced the
sustained release of prolactin in rats, as judged by deci-

duoma formation, and also blocked ovulation, indicating

l3

interference with LH release (Coppola §£.§£-o 1966). In
more recent studies, Coppola gt a1. (1965) also reported
that reserpine induced pseudopregnancy and attributed this
to depletion of brain norepinephrine stores. Kanematsu
£3 31. (1963) reported the site of action of reserpine to
be the basal tuberal hypothalamic area. This area of the
hypothalamus is considered to be the most important in rela—
tion to control of the anterior pituitary function (Flerko,
1966). Ratner 35 El- (1965) demonstrated that reserpine
can deplete rat hypothalamus of PIF activity when adminis-
tered in yiyg and can act directly on the hypothalamus
in_yitgg to decrease PIF release. Van Maanen and Smelik
(1968) provided direct evidence for localization of a mono-
aminergic prolactin inhibitory system in the tuberal part
of the hypothalamus. They observed induction of pseudo-
pregnancy in those rats implanted with reserpine and
blockage of pseudopregnancy in those rats pretreated with
iproniazid. It appears therefore, that sympathetic neuro-
transmitters may act on the nerve endings in the hypo-
thalamus containing the releasing and inhibiting factors,
to control their release into the hypophyseal portal vessels.
The above evidence has been further substantiated by
the work of Lu gt a1, (I970) in which serum and pituitary
prolactin levels were measured after administration of
reserpine, Chlorpromazine” d-methyl—meta—tyrosine and

0-methyl-para-tyrosine. These are all catecholamine

l4

depressants. All drugs produced increases in serum prolac-
tin levels indicating that they decreased hypothalamic PIF
activity. It is interesting to note that systemic injec-
tions of catecholamines such as epinephrine and dopamine
produced no effect on prolactin release (Lu gt al., 1970).
Koch §t_§l, (1970) has shown by an ig_yit£g study that the
effects of catecholamines on prolactin release may be dose
dependent. Kamberi gt El- (1971) perfused the anterior
pituitary with catecholamines and observed no effect on
prolactin release. However, when these same catecholamines
were injected into the third ventricle of the hypothalamus,
prolactin release was decreased. This indicates that cate-
cholamines act indirectly through the hypothalamic-hypo—

physeal system to influence prolactin secretion.

IV. Iproniazid

 

Monoamine oxidase (MAO) inhibitors block the enzyme,
monoamine oxidase which catalyses the oxidative deamination
of epinephrine, norepinephrine, dOpamine, serotonin and
others. The result is an accumulation of these amines in
the brain. One such MAO inhibitor is iproniazid which is a
"psychic energizer". Iproniazid was developed as a treat-
ment for tuberculosis but because of its excessive stimu-
lating effects on the CNS and its toxic effects, it was

discarded as an anti—tuberculosis drug (Bernheim, 1965).

15

Iproniazid has been used in order to study changes in
gonadotropic secretion since it is known that drugs of
this class alter catecholamine levels. Setnikar gt a1.
(1960) showed that iproniazid retards sexual maturation,
lengthens the estrous cycle, and causes an increase in
gonadotropic activity of the rat hypophysis. Poulson 23 a1.
(1960) demonstrated that iproniazid interrupted pregnancy
when administered before mid—pregnancy. In mice iproniazid
prevented the formation of decidual tissue (Lindsay gt al.,
1963). This evidence suggested that iproniazid alters
gonadotropic and possibly prolactin secretion. Mizuno §§_§l,
(1964) reported that iproniazid also may inhibit prolactin
release. They observed that the mammary secretory response
following electrical stimulation of the uterine cervix and
also postpartum lactation in rats were suppressed by iproni-
azid treatment. Nagasawa and Meites (1970) demonstrated
that iproniazid suppressed carcinogen-induced mammary tumor
growth in rats. This suggests a decrease in prolactin
secretion. Moreover, Lu and Meites (1971) demonstrated that
a systemic injection of iproniazid reduces serum prolactin
levels in female rats. It was of interest to determine if
iproniazid could inhibit the late afternoon proestrous surge

of prolactin.

16

V. Cocaine

Cocaine produces effects similar to stimulation of
adrenergic nerves and is an alkaloid obtained from leaves
of the coca tree. It paralyzes the termination of the
sensory nerves which is the basis for its therapeutic use
(Grollman, 1965).

In a review on drugs which affect norepinephrine up-
take, a hypothesis was proposed to explain the sympatho-
mimetic action of cocaine. It was stated that cocaine acts
by inhibiting the normally rapid inactivation of catechol-
amines by nerve uptake. When the uptake system is blocked
high concentrations of norepinephrine or other catechole
amines are available in the vicinity of adrenergic receptors,
this produces exaggerated and prolonged pharmacological
responses (Iversen, 1967). Direct evidence for an inhibi-
tion of norepinephrine uptake by cocaine was provided by
Whitby gt a1. (1960) and by Muscholl (1961). Cocaine was
administered intravenously followed by an intravenous injec—
tion of tritum labelled norepinephrine. It was observed
that cocaine markedly reduced the uptake of circulating nor—
epinephrine into certain tissues as measured by increased
plasma levels of norepinephrine (Whitby §t_§l,, 1960).

This work has been confirmed by various in giggg studies.
Dengler gt_§l, (1961) incubated cat cortex, spleen, and heart
tissues for 45 minutes in a medium containing cocaine and

labelled norepinephrine. Cocaine was observed to inhibit

17

norepinephrine uptake. Similar studies were performed by
Thoenen 33 El, (1964) and Gillespie and Kirpekar (1965) in
which they also demonstrated an inhibition of norepinephrine
uptake by cocaine. Fuxe and Hokfelt (1969) suggested that
cocaine could be used to study the relation of catechol-
amines to endocrine functions. Since no work had been done
on the relation of cocaine to anterior pituitary hormone
secretions until this time, it was of interest to determine

its effects on prolactin secretion.

VI. Barbiturates

Another class of drugs unrelated to catecholamines that
influence prolactin secretion are the barbiturates. The
barbiturates are derivatives of barbituric acid which is a
combination of urea and malonic acid. These drugs depress
the central nervous system and induce sedative and.hypnotic
effects. The sedatives and hypnotics are used to allay
nervousness, to induce Sleep if pain is absent and to control
convulsions (Grollman, 1965).

The barbiturates were first shown to influence gonado-
tropic secretion by Everett and Sawyer (1949). They injected
Nembutal into rats and were able to block ovulation in spon-
taneously ovulating animals. Further work by the same
authors showed that other barbiturates, such as phenobarbital
and barbitaI,ralso blocked ovulation (1950). Recently, with

the development of radioimmunoassays, work has been done in

18

which serum levels of hormones have been measured after
administration of particular drugs. Wuttke and Meites
(1970) reported that Na pentobarbital initially increases
and then depresses serum prolactin levels. Wakabayashi and
Arimura (1971) confirmed this report. In this thesis
further work was done to elucidate the site of action of Na
pentobarbital.

Thalidomide is structurally related to phenobarbital
and possesses prOperties similar to the barbiturates
(Grollman, 1965). It is a central nervous system depressant
but in contrast to barbiturates it does not cause the loss
of the righting reflex. It acts in conjunction with other
drugs to potentiate their effects such as potentiating the
depressant effect of Chlorpromazine (Kuhn and Van Maanen,
1961). It has been used widely as a sedative and mild .
hypnotic drug until it was discovered that thalidomide has
teratogenic effects (Grollman, 1965).

Since the discovery of its teratogenic properties,
comprehensive studies haVe been done on thalidomide and much
of the work has included tumor research. In 1966 it was re—
ported that thalidomide influenced the rate of growth and
disappearance of DMBA-induced tumors in Sprague-Dawley rats
(Muckter, 1966). Subsequent work by the same researcher and
co-workers showed that thalidomide derivatives, cyclic
imides, had more effect than thalidomide on DMBA induced

tumors in rats (Muckter gt al., 1967). More recently they

19

have reported that 1-(Morphalinomethyl)-4-phthalimido-
piperidendione-2,6, a thalidomide derivative, had a greater
therapeutic effect than bilateral ovariectomy, estrogen or
androgen on DMBA induced tumors (Muckter gt_§l., 1969).
They proposed that thalidomide and its derivatives exert
their effects on DMBA—induced tumors through the endocrine
system. It was of interest to know how thalidomide affects
prolactin secretion since it has been well established that
prolactin is influential in mammary tumorigenesis (Meites

and Nicoll, 1966).

VII. Ergot Drugs

Ergot alkaloids are derivatives of lysergic acid and
are extracted from a rye or wheat fungus, ergot. Thera-
peutically ergot alkaloids were used to alleviate headaches
due to their vasoconstrictor activity. They also cause
smooth muscle contraction and are adrenergic blocking agents
(Grollman, 1965).

Shelesnyak (1954, 1955) showed that ergotoxine inhibited
deciduoma formation and terminated pseudopregnancy and early
pregnancy. This suggested that ergotoxine interrupted pro-
~1actin secretion. In subsequent work it was demonstrated
that exogenous prolactin was able to overcome the effects
of ergotoxine on interrupting gestation (Shelesnyak, 1958).
Zeilmaker and Carlsen (1962) reported that ergocornine pro-

duced morphological changes in the corpora lutea which

20

prohibited decidual formation and inhibited milk production
in lactating rats, and this was overcome by an injection of
lactOgenic hormone. Nagasawa and Meites (1970) reported

that ergocornine reduced serum and pituitary prolactin

levels and suppressed DMBA-induced mammary tumor growth when
injected for 15 days. A systemic injection or an implant
into the median eminence area of the hypothalamus of ergo—
cornine significantly depressed cyclic fluctuations in serum
prolactin levels (Wuttke £3 31., 1971). Wuttke £3 31. (1971)
also reported that part of the inhibitory action of ergo—
cornine on prolactin secretion was mediated through the hypo-
thalamus by increasing PIF activity. In_yit£g studies by

Lu §t_§l, (1971) have shown that ergocornine also can act
directly on the anterior pituitary to inhibit prolactin
secretion. Further evidence will be presented in this thesis

that ergocornine inhibits prolactin release.

VIII. Effects of Hormones

Prolactin is also influenced by other hormones as well
as various drugs. It has been demonstrated that administra-
tion of gonadal, adrenal cortical, and thyroid hormones
increased prolactin secretion by the in_§itu anterior pitui-
tary of rats (Meites 33 31., I963). Estrogen is the most
potent stimulator of prolactin release. Nicoll and Meites
(1962) added estrogens (0.5 ug per ml) to the medium of organ

cultures of rat anterior pituitaries and found a significant

21

increase in prolactin production in yitgg, Subsequent work
by Nicoll and Meites (1964) showed that lower doses of
estrogen are more effective in stimulating prolactin pro-
duction ifl.!l££2 than are higher doses of estrogen.

In an in yiyg study, Ramirez and McCann (1964) implanted
estrogen tipped needles into the anterior pituitary and
median eminence of rats, and observed that the intrahypo-
thalamic and intrahypophyseal implants promoted pseudo-
pregnancy, mammary growth, and lactation. This confirmed
the in_yit£9 findings that estrogen acts directly on the
anterior pituitary to stimulate prolactin secretion. Ratner
and Meites (1962) demonstrated that the anterior pituitary
in estrogen-injected rats released greater amounts of pro-
lactin than the pituitary Of untreated rats during a short
time incubation. More recently Nagasawa £2.21- (1969) showed
that an implant of estradiol benzoate into the median
eminence of rats significantly increased serum and pituitary
prolactin levels. Therefore estrogen has two modes of action
in releasing prolactin, one by a direct action on the pitui-
tary and the other indirectly through the hypothalamus.

Other steroids such as progesterone, androgens, and
corticosteroids slightly stimulate prolactin secretion
in giggg (Meites, 1959), but were observed to be without
effect on prolactin secretion ig_yitgg_(Nicoll and Meites,
1966). The thyroid hormones also influence prolactin

secretion. Injections of thyroid hormones in rats increased

. _..._-—:. iilL. 1"
" H A A I e

 

 

22

prolactin secretion by the in situ pituitary (Meites, 1963).

.Nicoll and Meites (1963) further demonstrated that addition

of either thyroxine or triiodothyronine to the medium of
organ cultures of rat anterior pituitary resulted in a sig-
nificant stimulation of prolactin release. Thus a direct
action of thyroid hormones on the anterior pituitary was

established.

MATERIALS AND METHODS

I. Animals

All rats were of the Sprague-Dawley strain obtained
from two sources: Spartan Research Labs. (Haslett,
Michigan) and Carworth Farms (Portage, Michigan). Female
and male rats were used and housed 6 or 8 to a cage. After
drug treatment was started the animals were separated into
different groups. All animals were fed Wayne Lab Blox
pellets (Allied Mills, Chicago, Illinois) and water ad lib;—
33m, The temperature of the animal room was maintained at
25:10C and artificially illuminated with 14 hours of light

(5:00 AM to 7:00 PM) and 10 hours of darkness daily.

II. Treatment of Pituitary and Blood Samples

All blood samples were taken by cardiac puncture under
light ether anesthesia and pituitaries were removed at the
end of each experiment. The pituitaries were frozen at
-20°C until assayed, at which time pituitary homogenates
were made. The pituitaries were sonified with a Sonifier
cell disruptor (Heat Systems-Ultrasonics, Inc., Plainview,

N.Y.) in a tube containing 0.01 M phosphate buffered saline

23

24
(pH 7.0). The blood samples were first stored for 24 hours
at 2.5°C and the clots were then removed. The sera were

frozen at —20°C until assayed.

III. Radioimmunoassay

 

r.
Prolactin in individual serum samples and pituitaries h?

were measured by radioimmunoassay (Niswender gt $1., 1969).

Each serum sample was assayed at 2 dilutions and pituitary

 

samples at 3 and 4 dilutions in 1%.bovine serum albumin and j

1% egg white. These values were averaged and expressed in
terms of a standard rat prolactin preparation (NIAMD-RP-l)
obtained from National Institutes of Arthritis and Metabolic
Diseases of National Institutes of Health, Bethesda, Mary-
land. The hormone (H-lO-lO-B) used for iodination in these
assays was provided by Dr. S. Ellis, NASA, Ames Research
Center, Moffet Field, California. Part of the radioimmuno-
assays done utilized iodine 131 (Cambridge Nuclear, Bil-
lerica, Mass.) for iodination of the hormone and in the last
two experiments (cocaine and thalidomide) the isotope,
iodine 125 (New England Nuclear, Boston, Mass.) was used for

labelling of the hormone.

IV. Histological Treatment

Ovarian tissue was fixed in Bouin's fluid, sectioned
and stained with hematoxylin and eosin and examined under

the microscope.

25

V. Statistical Treatment

Sample means and standard error of means were calcu—
lated within each experimental group and significance of
differences between groups was determined by Student's t

test and Analysis of Variance (Sokal and Rohlf, 1969).

EXPERIMENTAL

I. Effects of Barbiturates on Prolactin Secretion

F

A. Effect of Injecting Na Pentobarbital (PB) on :3
Hypothalamic PIF Activity and Effect of Na 5
Pentobarbital on Pituitary Prolactin Release
In Vitro in the Rat

 

1. Objective éi I

Recently it was reported that an injection of PB dur- i
ing the early afternoon of proestrus produced an initial
increase in serum prolactin for about 30 minutes duration,
but completely prevented the late afternoon rise in serum
prolactin (Wuttke and Meites, 1970). The purpose of this
experiment was to determine whether PB acted on the hypo—
thalamus, on the anterior pituitary or on both to influence

prolactin release.

2. Procedure

Adult female (160-200 g) and male (180-220 9) rats were
used. Thirty female rats were divided int03 groups and
treated at 1 PM on the day of proestrus as follows:

Ten rats were injected intraperitoneally (i.p.) with
31.5 mg NaPB base in .9% saline/kg BW and guillotined 10

minutes later. Ten other animals were injected i.p. with

26

27

31.5 mg PB/kg and guillotined 2 hours later. Ten control
animals were injected i.p. with .9%)sa1ine and guillotined
2 hours later.

Immediately after each rat was killed, the hypothalamus
and an equivalent amount of cerebral cortex were removed.
The hypothalamic and cerebral cortical tissues from each
group were placed in ice cold .1 N HCl, homogenized at
12,000 g for 40 minutes at 4°C. The supernatant was kept
frozen, thawed just prior to use, adjusted to pH 7.4 with
1 N NaOH and assayed for PIF activity by the ig_yi§£g_method
of Kragt and Meites (1965).' Donor anterior pituitaries
were removed from mature male rats, hemisected, and each
half was incubated separately in.l ml medium 199 with .1
m1 of hypothalamic or cerebral cortical extract (equivalent
to approximately 1 hypothalamus). Incubation time was 4
hours.

In order to test the direct effect of PB on the anterior
pituitary, 10 anterior pituitary halves from male rats were
each incubated separately in medium 199 without brain ex-
tracts, and the corresponding 10 halves were each incubated
in medium 199 to which 3 ug PB was added. Incubation time

was 4 hours.

3. Results
Pituitary halves incubated with hypothalamic extract

from saline—injected control rats (Group A1) released

28

significantly less prolactin into the medium than the corre-
sponding halves incubated with cerebral cortical extract

from the same rats (Group A2), indicating the presence of

PIF activity in the hypothalamus (Table 1). Ten minutes
after PB injection, no significant PIF activity was found in
the hypothalamus, as indicated by release of similar amounts
of prolactin from AP halves incubated with either hypothala-
mic or cerebral cortical extract. Two hours after PB in-
jection, hypothalamic PIF activity was partially restored,

as indicated by lower prolactin release from pituitary halves
incubated with hypothalamic extract than by the corresponding
halves incubated with cerebral cortical extract. This shows
that PB decreases hypothalamic PIF activity, and is be-
lieved to account for the initial rise in serum prolactin
after injection of PB.

Pituitary halves removed from untreated male rats and
incubated without brain extracts (Group Bl) released sig-
nificantly more prolactin than the corresponding halves
incubated with 3 ug PB (Group B2). This indicates that PB
directly inhibits AP prolactin release, and is believed to

account for the inhibition of prolactin release by PB.

 

29

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spfisfipoe mHm seamfimnpoasm co Ammv HmpfinanOpcmm waspomncH co somecm .<.H «Hams

30

B. Effects of Thalidomide on Serum and
PituitaryfiProlactin Levels in the
Rat

1. Objective

 

The purpose of this study was to determine the effects
of thalidomide on serum and pituitary prolactin levels and
relate these findings to the effects of other barbiturates

on prolactin release.

2. Procedure

Adult female rats weighing between 210 and 245 grams
were used. A pre-treatment blood sample was taken at 12 AM
on the day of proestrus from each rat. A dose of 5, 10,
or 25 mg thalidomide/100 gm body weight was injected i-p. at
12:30 AM and rats were bled at l, 2, 3, 4, and 5 PM. Control
rats were injected with saline, the drug vehicle. The ani-
mals were guillotined after the 5 PM bleeding and their
pituitaries were removed. Both pituitary and serum samples

were assayed for prolactin by radioimmunoassay.

3. Results

Table 2 shows that a single i.p. injection of any of
the 3 doses of thalidomide significantly reduced serum pro—
lactin. The afternoon proestrous surge was completely
blocked. A dose-response effect ofthalidomide is indicated,
since the highest dose of thalidomide used (25 mg/100 g
body weight) produced the greatest blockage of the proestrous

prolactin peak, whereas serum prolactin levels were

1..
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.m mHowB

increasingly higher when lower doses of thalidomide (5 and
10 mg/100 g body weight) were used. The serum prolactin
levels appeared to be reflected by the pituitary prolactin
concentrations. Thus pituitary concentrations were sig-
nificantly higher in all animals treated with thalidomide.
This might be expected since the release of prolactin from

the pituitary was prevented by the drug.

C. Conclusions

Na pentobarbital produced a disappearance of hypothal-
amic PIF activity by 10 minutes after injection and depressed
PIF activity after 2 hours. Cerebral cortical extract from
the same animals had no effect on pituitary prolactin re—
lease ig_yit£9. Incorporation of 3 ug PB directly into the
incubation medium significantly inhibited prolactin release
by a direct action on the anterior pituitary. These results
suggest that the ability of injected PB to initially increase
prolactin release for a period of about 30 minutes and then
to inhibit prolactin release for a more prolonged period
(Wuttke and Meites, 1970) is due to a rapid reduction in
hypothalamic PIF activity followed by a prolonged inhibitory
action directly on the pituitary.

Thalidomide, like PB, decreased serum prolactin levels
and blocked the afternoon proestrous rise. Thalidomide
also increased pituitary prolactin concentration as compared

to that in control rats. However, thalidomide unlike PB,

33

did not cause an initial increase in serum prolactin.

This may be due to the fact that the doses of thalidomide
used did not completely anesthetize the animals as did PB.
Whether thalidomide, like PB, directly blocks pituitary

release of prolactin remains to be determined.

II. Effects of Drugs that Alter Hypothalamic
Catecholamines on Prolactin Release

A. Effects ofga Single Injection of Iproniazid
on Pituitary Prolactin Release
1. Objective
Iproniazid has been shown to inhibit prolactin release
on the morning of proestrus in female rats (Lu and Meites,
1971). It was of interest to determine if iproniazid would

block the proestrous afternoon rise in prolactin as well.

2. Procedure

 

Adult female rats weighing between 205 and 245 grams
were used. A pre-treatment blood sample was taken at 1 PM
on the day of proestrus. The animals were injected with a
dose of 40 mg/rat of iproniazid at 1:15 PM and bled at 2,

3, and 4 PM. The animals were killed by guillotine. The
control animals were injected with saline, the drug vehicle.
Serum samples were assayed for prolactin by radioimmuno-

assay.

34

3. Results

It is evident from the data shown (Table 3) that
iproniazid blocked the proestrous afternoon peak. The
action of iproniazid was seen approximately 45 minutes after
injection (1 hr group). At this time there was no increase
in prolactin release as compared to the control rats. When
the control animals reached their peak level (1200:100.4
ng/ml) of prolactin at 4 PM, the iproniazid group showed no
significant rise in serum prolactin. Since iproniazid is a
monoamine oxidase inhibitor, and thus inhibits the meta-
bolism of catecholamines, it acts to increase catecholamine
activity in the brain (Grollman, 1965). An increase in
catecholamine activity has been shown to evoke an increased
release of PIF by the hypothalamus (Lu and Meites, 1971)

and depress prolactin release.

B. Effects of Cocaine on Pituitary and
Serum Prolactin Levels
1. Objective
Cocaine is a weak monoamine oxidase inhibitor. More
importantly it potentiates the action of catecholamines.
Since it has been shown that catecholamines inhibit pro-
lactin release, it was of interest to determine the effects

of cocaine on serum prolactin values.

2. Procedure
Adult female rats weighing between 210 and 260 grams

were used. A pre-treatment blood sample was taken at 9 AM

35

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.m oHDME

36

on the morning of proestrus and the last sample was taken
before the proestrous afternoon rise of prolactin. A dose
of l, 5, 10, or 20 mg/kg was injected i.p. at 9:15 AM and
rats were bled 30 minutes, one hour, two hours, and four
hours after injection. Control rats were injected with
saline, the drug vehicle. The animals were guillotined

at the end of the experiment and their pituitaries were
removed and weighed. Both pituitary and serum samples were

assayed for prolactin by radioimmunoassay.

3. Results

The data (Table 4) shows that cocaine elicited a de-
crease in serum prolactin levels. The lower doses (1 and
5 mg/kg) lowered serum prolactin levels below the pre-
injection values. However, the action of the higher doses
(10 and 20 mg/kg) appeared to be less consistent. The 10
mg dose evoked a significant reduction in serum prolactin
0.5 hour after injection, followed by an increase at l and
2 hours after injection and then a fall at 4 hours below
pre-treatment values. The dose of 20 mg/kg appeared to be
more effective in reducing serum prolactin levels, but after
4 hours there was a significant increase in serum prolactin.
None of the doses used had any effect on pituitary prolac-

tin concentration by the end of 4 hours.

 

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38

C. Conclusions

 

Both iproniazid and coCaine have been reported to
increase catecholamines in the brain and other tissues
(Iversen, 1967), although they do not accomplish it in the
same way. As mentioned earlier iproniazid blocks the en—
zyme, monoamine oxidase which catalyzes the oxidative
deamination of epinephrine, norepinephrine, dopamine, and
others, whereas cocaine blocks the reuptake of catecholamines
by the nerve endings. Both processes result in increased
levels of catecholamines in specific tissues. Increases in
hypothalamic catecholamines have been shown to be associated
with increased hypothalamic PIF activity and decreased
serum prolactin values (Lu and Meites, 1971). Cocaine ap-
peared to produce a biphasic effect on prolactin release.
The higher doses (10 and 20 mg/kg) caused both an inhibition
and stimulation of prolactin release. It is possible that
the biphasic effect on prolactin release is due to other
actions of cocaine besides its ability to potentiate the
actions of catecholamines. It is known that high doses of
cocaine cause a heightened excitability in animals (Grollman,
1965) and this may stimulate prolactin release. There are
many indications that stresses increase prolactin release
(Meites gt gl., 1963). It remains to be determined whether
these drugs act only via the hypothalamus or also on the

pituitary.

39

III. Luteolytic Role of Prolactin During the
Estrous Cycle in Female Rats

A. Effects of Ergocornine (EC) Treatment

1. Objective

 

Recently it was reported that injections of ergocornine,
at a dose known to markedly reduce serum prolactin during
the estrous cycle prevented luteolysis of old corpora lutea
(Wuttke and Meites, 1971). Luteolysis of corpora lutea
during the estrous cycle of the rat has generally been
ascribed to LH (Rothchild, 1965). It was of interest to re—
peat some of the work previously reported (Wuttke and
Meites, 1971), using higher doses of ergocornine known to
reduce serum LH as well as prolactin levels to determine
the effects of reducing the levels of both hormones on luteal

maintenance.

2. Procedure

 

Adult female rats weighing 205 to 245 grams were used.
The animals were injected at 9 AM and 6 PM daily with ergo-
cornine methanesulfonate (EC), 400 ug and 800 ug/rat i.p.,
dissolved in 70% ethanol and then in .9% saline to give a
solution of 1% ethanol - 99% saline. The treatment was
started on the last afternoon of diestrus prior to the ex-
pected day of proestrus and continued through the cycle
until the following first day of diestrus. Control animals

were injected with the ethanol—saline solution. At 2 PM on

 

40

the day of proestrus, 8 of the 16 rats given each dose of
ergocornine, received a subcutaneous injection of 1 mg pro-
lactin (ovine, NIH-P-S8, 28 IU/mg) dissolved in .9% saline.
At the end of treatment, during diestrus, the rats were
killed and the ovaries, adrenals, and uteri were removed,
cleaned and weighed. The ovaries were prepared for histo-
logical examination. Corpora lutea were counted only from a

mid-sagittal cross section and not from the entire ovary.

3. Results

Injections of both doses of ergocornine (400 ng and
800 ug/rat) resulted in significant increases in both
ovarian and adrenal weight, and no effect on uterine weight
as compared to controls (Table 5). The number of corpora
lutea per ovary in the EC-treated rats was significantly
greater than the corpora lutea per ovary in the controls
or rats given both EC and prolactin. It was noted during
microsc0pic examination that many of the ovaries of the EC-
treated animals had large numbers of follicles. Body weight
was significantly although not greatly reduced in the EC—
injected rats as compared to body weight in the control
rats. Thus the large doses of EC prevented luteolysis of
the corp0ra lutea and prolactin counteracted the effect of

the EC on the ovaries.

 

P.» -I

 

41

 

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va mcHCLooome
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42

B. Effects of Iproniazid Treatment

1. Objective

It has been reported in this thesis that iproniazid
decreases serum prolactin levels and also blocks the pro—
estrous afternoon,surgeoofnprolactin. It was of interest
therefore to see whether iproniazid could be used to pro-
vide evidence for a luteolytic role of prolactin during the

estrous cycle, since it should decrease prolactin release.

2. Procedure

 

Adult female rats weighing 200 to 240 grams were used.
The animals were divided into four groups as follows:
(1) saline controls, treated with the drug vehicle, (2) ani-
mals given 10 mg of iproniazid daily, and 45 mg on the day
of proestrus, (3) iproniazid treated animals given 5 mg of
iproniazid daily and 25 mg on the day of proestrus, and
(4) iproniazid as in group 2, and at 2 PM on the afternoon
of proestrus 1 mg of prolactin (ovine, NIH-P—S8, 28 IU/mg)
dissolved in saline. The treatments were started on the
last PM of diestrus prior to the expected day of proestrus
and continuing throughout one cycle, terminating on the next
first day of diestrus. In another group of rats, this same
schedule and grouping of animals was carried out for three
cycles as well. However, in the three cycle study, only
one group was treated with iproniazid alone (same doses as
group 3 in the one cycle study). At the end of the treat-

ment, during diestrus, the rats were killed and the ovaries

 

43

and pituitaries were removed. At the end of the three
cycle experiment, the adrenals and uteri were also

removed. The ovaries were cross sectioned and prepared for
microscopic examination. Corpora lutea were counted only

from the cross section and not from the whole ovary.

3. Results

The results obtained with iproniazid in the one cycle
study were very similar to those reported for ergocornine
(Wuttke and Meites, 1971). Injections of iproniazid (the
two dose groups) resulted in a significant increase in
ovarian weight as compared to the controls (Table 6).

The ovaries of rats injected with iproniazid and prolactin
weighed about the same as in control rats. The pituitaries
of one iproniazid treated group (Group 2) and iproniazid

and prolactin treated animals (Group 4) were significantly
decreased as compared to the controls. The number of

corpora lutea per ovarian cross section in the iproniazid
treated rats was significantly greater than the corpora

lutea per ovary in the controls or rats given both iproniazid
and prolactin.

The effects of iproniazid treatment during three
estrous cycles (Table 7) resembled those during one cycle.
The ovaries on the iproniazid treated animals were signifi-
cantly increased as compared to the controls or rats given
both iproniazid and prolactin. The number of corpora lutea

per cross section of ovary were increased in weight in the

Jr,

44

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45

 

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46

rats given iproniazid alone, and returned to normal control
numbers in the rats given iproniazid together with pro-
~1actin. There was no significant differences in uterine

or pituitary weights in the iproniazid-treated rats as
compared to controls in the three cycle study (Table 8).
However, the average weight of the adrenals was significantly
decreased in both the iproniazid and iproniazid prolactin
treated animals as compared to the controls. In both the

one cycle and three cycle studies iproniazid caused a slight
decrease in body weight but this was not significant.

It should be noted that in the three cycle experiment
the dosage of iproniazid was reduced (5 mg daily and 25 mg
on day of proestrus to 5 mg daily and 10 on day of proestrus)
in the middle of the experiment because of the overexcita-
bility of the animals. Many large follicles were observed

in the ovaries.

C. Conclusions

 

The data presented here provide further evidence that
the function of prolactin during the estrous cycle is to
induce luteolysis of the previous crop of corpora lutea.
Both iproniazid and ergocornine significantly increased
the weights of the ovaries and number of corpora lutea as
compared to the controls. Prolactin administration pre-
vented these actions of the 2 drugs. Although neither serum

prolactin nor LH were measured in these rats, both drugs

""1

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47

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48

have been shown to significantly decrease serum prolactin
levels, and the high doses of ergocornine used were also
shown to decrease serum LH values (Wuttke §t_gl., 1971).
It does not appear therefore, that LH has any role in
luteolysis of the old corpora lutea and that prolactin is

solely responsible for this action during the estrous cycle.

 

DISCUSSION

It was the purpose of this thesis to use drugs to
learn more about the control of prolactin secretion and
the function of prolactin in the cycling animal. The drugs
used in these experiments all depress serum prolactin
levels. .However, pentobarbital (Wuttke and Meites, 1970)
has the ability to both stimulate and inhibit prolactin
release, and cocaine may have the same properties. Two
sites of action that these drugs may utilize to produce
their effects on prolactin release are the hypothalamus and
the pituitary.

As reported here, pentobarbital has a dual action. It
depletes the hypothalamus of PIF activity and acts directly
on the pituitary to inhibit prolactin release. This is be-
lieved to explain its ability to first stimulate prolactin
release by depressing hypothalamic PIF activity, and then
to inhibit prolactin release by a direct inhibitory effect
on the pituitary. Ergocornine has also been shown to act
on both the hypothalamus and the pituitary to inhibit pro-
lactin release (Wuttke gt él-I 1971; Lu gt gl., 1971).
Ergocornine increases PIF activity in the hypothalamus whereas
pentobarbital decreases PIF activity in the hypothalamus, but

both inhibit the pituitary directly.

49

if;

50

The site of action of thalidomide has not yet been
determined. Since it is a barbituric acid derivative, it
may act similarly to pentobarbital. Apparently, it does
not produce an initial increase in serum prolactin levels
like pentobarbital, perhaps because it has less anesthetic

effect. Its action may be solely on the pituitary.

 

nI.
Thalidomide has been shown experimentally to act differently ?
from the barbiturates in many respects and therefore a
direct comparison can not be made between the 2 drugs "
(Somers, 1960). Unlike the barbiturate drugs, thalidomide vi ‘

does not cause an initial excitation in mice, incoordination,
respiratory depression or narcosis, and it is virtually non-
toxic.

Iproniazid and cocaine influence catecholamine levels
in the hypothalamus. Iproniazid, a monoamine oxidase in—
hibitor depresses the metabolism of catecholamines and there-
by increases catecholamine concentration in the hypothalamus
(Grollman, 1965). Cocaine, on the other hand, blocks the
reuptake of catecholamines by nerve endings so that the
concentration of these amines is also increased in the hypo-
thalamus (Iverson, 1967). The data presented reaffirms the
hypothesis by Coppola §£.§l- (1966) and others that an
increaSed level of catecholamines inhibits prolactin and
stimulates LH and FSH release. Kamberi gt a1. (1971)
demonstrated that dopamine Causes an increase in the release
of PIF by diencephalic elements into the hypothalamo-

pituitary portal vessels and thereby inhibits prolactin

 

51

release. It would prove interesting to measure catechol—
amine concentrations or turnover rates of catecholamines in
the hypothalamus after administration of pentobarbital,
ergocornine, and thalidomide to determine if there are any
correlations with prolactin release.

The function of prolactin during the estrous cycle was

not known until recently. Some of the data presented in 53

this thesis provide further evidence for the action of pro- :17
lactin as a luteolytic agent during the estrous cycle. As f I
reported by Wuttke and Meites (1971), prolactin causes 3 j

regression of the old crop of corpora lutea from the previous
cycle. If the normal action of prolactin during proestrus
and estrus is inhibited, by either ergocornine or iproniazid,
ovarian weight increases due to the accumulation of corpora
lutea. Injections of prolactin into ergocornine or iproni-
azid treated animals once during each cycle induced atropy

of the old corpora lutea. Billeter and Fluckiger (1971)
demonstrated a luteotropic as well as a luteolytic action

of prolactin in the cycling rat. They used another ergot
derivative, ergokryptine, to inhibit prolactin. However,
instead of injecting prolactin only on the days of proestrus
and estrus (Wuttke and Meites, 1971), they injected prolactin
every day for 21 days. Some of their rats given ergokryptine
and prolactin failed to ovulate and maintained the corpora
lutea. Their failure to separate the luteotropic from the

luteolytic effects of prolactin makes their study less valid

 

52

than that reported by Wuttke and Meites (1971) and the
experiments presented here.

It is evident that prolactin can act as either a luteo-
lytic or luteotropic agent. According to Malven (1969) the
action of prolactin depends on the time of its administra-
tion after corpus luteum formation. An intriguing question
which arises from these studies is——what is the mechanism
which causes prolactin to function as either a luteotropic
or luteolytic agent? The answer appears to lie in the ovary
itself, since it has been shown that prolactin acts on fresh
corpora lutea to elicit progesterone secretion but on old
corpora lutea to induce luteolysis (Rothchild, 1965; Malven
and Sawyer, 1966). It is well established that during preg-
nancy in rats prolactin is necessary to maintain the corpora
lutea (Clemens §t_§l,, 1969). It has been reported that
prolactin inhibits the enzyme, 20 a-hydroxysteroid dehydro—
genase, which converts progesterone to 20 d—dehydroproges-
terone, a less active compound (Wiest §t_§l,, 1968). This
possibility has been discussed by Malven (1969). Apparently

prolactin does not have this action on old corpora.lutea,

and induces luteolysis. The precise biochemical mechanism(s)

involved in luteolysis remains to be determined.

LIST OF REFERENCES

LIST OF REFERENCES

Amenomori, Y., C. L. Chen, and J. Meites. 1970. Serum
prolactin levels in rats during different reproductive
states. Endocrinology 86: 506-510.

Astwood, E. B. 1941. The regulation of corpus luteum func-
tion by hypophysial luteotrophin. Endocrinology 28:
309-320.

Barraclough, C. A., and C. H. Sawyer. 1959. Induction of
pseudopregnancy in the rat by reserpine and chlorpro-
mazine. Endocrinology 65: 563-579.

Ben-David, M. 1968. The role of the ovaries in perphen-
azine-induced lactation.‘ J. Endocrinol. 41: 377-385.

Bern, H. A., and C. S. Nicoll. 1968. The comparative endo-
crinology of prolactin. Recent Progr. in Horm. Res.
24: 681-720.

Bernheim, F. 1965. Chemotherapy of bacterial infections:
Drugs used in treatment of tuberculosis. 13: Drill's
Pharmacology in Medicine. J. R. DiPalma (Ed.). McGraw-
Hill, New York. 1341—1342.

Billeter, E., and E. Fluckiger. 1971. Evidence for a luteo-
lytic function of prolactin in the intact cyclic rat
using 2-Br-a-ergokryptine (CB154). Experientia 27:
464—465.

Boler, J., F. Enzmann, K. Folkers, C. T. Bowers, and A. V.
Schally. 1969. The identity of chemical and hormonal
properties of the thyrotropin releasing hormone and
pyroglutamyl—histidyl—proline-amide. Biochem. Biophys.
Res. Comm. 37: 705-710.

Chen, C. L., Y. Amenomori, K. HZ Lu, J. L. Voogt, and J.
Meites. 1970. Serum prolactin levels in rats with
pituitary transplants or hypothalamic lesions. Neuro-
endrocrinology 6: 220—227.

Clemens, J. A., M. Sar and J. Meites. 1969. Termination of

pregnancy by prolactin implants in the median eminence
of the rat. Proc. Soc. Exp. Biol. Med. 130: 628-630.

53

54

Clifton, K. H., and J. Furth. 1960. Ducto-alveolar growth
in mammary glands of adreno—gonadectomized rats bearing
mammotropic pituitary tumors. Endocrinology 66: 893-
897. '

Coppola, J. A., R. G. Leonardi, W. Lippman, J. W. Perrine,
and I. Ringler. 1965. Induction of pseudopregnancy in
rats by depletors of endogenous catecholamines.
Endocrinology 77: 485—490.

Coppola, J. A., R. G. Leonardi, and W. Lippman. 1966. Ovula—
tory failure in rats after treatment with brain nor-
epinephrine depletors. Endocrinology 78: 225-228.

Coppola, J. A. 1971. Brain catecholamines and gonadotropin
secretion. lg: Frontiers in Neuroendocrinology. L.
Martini and W. F. Ganong (eds.). Oxford University
Press, New York. 129—143.

Cowie, A. T., and S. J. Folley. 1955. Physiology of the
gonadotropins and the lactogenic hormone. ‘13: The
Hormones, Vol. IV. G. Pincus and K. V. Themann (eds.),
Academic Press, New York. 309—387.

Dao, T. L. 1962. The role of ovarian hormones in initiating
the induction of mammary cancer in rats by polynuclear
hydrocarbons. Cancer Res. 22: 973—981.

Dengler, H. J., H. E. Spiegel, and E. O. Titus. 1961.
Effects of drugs on uptake of isotopic norepinephrine.
Nature 191: 816-817.

Elias, J. J. 1957. Cultivation of adult mouse mammary
glands in hormone-enriched synthetic medium. Science
126: 842-844.

Everett, J. W., and C. H. Sawyer. 1959. The blocking effect
of Nembutal on the ovulatory discharge of gonadotrophin
in the cyclic rat. Proc. Soc. Exp. Biol. Med.- 71:
696—698.

Everett, J. W., and C. H. Sawyer. 1950. A 24-hour perir
odicity in the "LH-release apparatus" of female rats
disclosed by barbiturate sedation. Endocrinology 47:
198-217.

Everett, J. W., 1956. Functional corpora lutea maintained
for months by autografts of rat hypophysis. Endo—
crinology 58: 786—796.

Flerko, Bela. 1966. Control of gonadotropin secretion in
the female. In: Neuroendocrinology. Vol. I. ‘L.
Martini and W. F. Ganong (eds.). Academic Press,

New York. 613-668.

55

Folley, S. J., and F. G. Young. 1941. Prolactin as a
specific lactogenic hormone. Lancet 240: 380-381.

Fuxe, K., and T. Hokfelt. 1969. Catecholamines in the
hypothalamus and the pituitary gland. In: Frontiers
in Neuroendocrinology. W. F. Ganong and L. Martini
(eds.). Oxford University Press, New York. 47-96.

Gay, V. L., A. R. Midgley, and G. D. Niswender. 1970.
Patterns of gonadotropin secretion associated with
ovulation. Fed. Proc. 29: 1880.

Gillespie, J. A., and S. M. Kirpekar. 1965. The inactiva—
tion of infused noradrenaline by the cat spleen.
J. Physiol. 176: 205-227.

Grollman, A. 1965. Pharmacology and Therapeutics. Sixth
Ed. Lea and Febiger, Philadelphia, Pa. 464-468;
335-343; 202—203.

Grosvenor, C. E., S. M. McCann, and M. D. Nallor. 1964.
Inhibition of suckling-induced release of prolactin
in rats injected with acid extract of bovine hypo-
thalamus. Program 46th Meeting Endocrine Soc.,

San Francisco, p. 46.

Huggins, C., G. Briziarelli, and H. Sutton. 1959. Rapid
induction of mammary carcinoma in the rat and the
influence of hormones on the tumors. J. Exp. Med.
109: 25-42.

Huggins, C., L. C. Grand, and F. P. Bullantes. 1961.
Mammary cancer induced by a single feeding of poly-
nuclear hydrocarbons and its suppression. Nature 189:
204—207. '

Huggins, C. 1965. Two principles in endocrine therapy of
cancer: hormone deprival and hormone interference.
Cancer Res. 25: 1163—1167.

Iversen, L. L. 1967. The uptake and storage of noradrenaline
in sympathetic nerves. Cambridge University Press,
London. 151—155.

Kamberi, I. A., R. S. Mical, and J. C. Porter. 1971. Effect
of anterior pituitary perfusion and intraventricular
injection of catecholamines on.prolactin release.
Endocrinology 88: 1012-1020.

Kanematsu, S., J. Hilliard, and C. H. Sawyer. 1963. Effect
of reserpine on pituitary prolactin content and its
hypothalamic site of action in the rabbit. Acta Endo-
crinologica 44: 467—474.

56

Kim, U., J. Furth, and K. H. Clifton. 1960. Relation of
mammary tumors to mammotropes. I. Induction of mammary
tumors in rats. II. Hormone responsiveness of 3-
methylcholanthrene-induced mammary carcinomas. III.
Hormone responsiveness of transplanted mammary tumors.
Proc. Soc. Exp. Biol. Med. 103: 640-650.

Koch, Y., K. H. Lu, and J. Meites. 1970. Biphasic effects
of catecholamines on pituitary prolactin release
in_vitro. Endocrinology 87: 673-675.

Kragt, C. L., and J. Meites. 1965. Stimulation of pigeon
pituitary prolactin release by pigeon hypothalamic
extract in vivo. Endocrinology 76: 1169-1176.

Kuhn, W. L., and E. F. Van Maanen. 1961. Central nervous
system effects of thalidomide. J. Pharmacol. Exp.
Therap. 134: 60—68.

Li, C. H., J. S. Dixon, K. D. Schmidt, Y. A. Pankov, and
T. B. Lo. 1969. Amino and carboxy-terminal sequences
of ovine lactogenic hormone. Nature 222: 1268—1269.

Lindsay, D., E. Poulson, and J. M. Robson. 1963. The
effects of 5-hydroxytryptamine on pregnancy. J. Endo-
crinol. 26: 85-96.

Lu, K. H., Y. Amenomori, C. L. Chen, and J. Meites. 1970.
Effects of central acting drugs on serum and pituitary
prolactin levels in rats. Endocrinology 87: 667-672.

Lu, K. H., Y. Koch and J. Meites. 1971. Direct inhibition
by ergocornine of pituitary prolactin release. Endo-
crinology 89: 229-233.

Lu, K..H., and J. Meites. 1971. Inhibition by l-dopa and
monoamine oxidase inhibitors of pituitary prolactin
release; stimulation by methyl-dOpa and d-amphetamine.
Proc. Soc. Exp. Biol. Med. 137: 480-483.

Lyons, W. R., C. H. Li, and R. E. Johnson. 1958. The hor-
monal control of mammary growth and lactation. Recent
Progr. Horm. Res. 14: 219-254.

Malven, P. V. 1969. Luteotrophic and luteolytic responses
to prolactin in hypophysectomized rats. Endocrinology
84: 1224-1229.

Malven, P. V., and C. H. Sawyer. 1966. A.luteolytic action
of prolactin in hypOphysectomized rats. Endocrinology
79: 268-274.

57

Matsuo, H., Y. Baba, R. M. G. Nair, A. Arimura, and A. V.
Schally. 1971. Structure of the procine LH- and FSH-
releasing hormone. I. The proposed amino acid sequence.
Biochem. Biophys. Res. Comm. 43: 1334-1339.

Meites, J. 1959. Mammary growth and lactation. In:
Reproduction in Domestic Animals, Vol. I. .H..H. Cole
and P. T. Cupps (eds.). Academic Press, New York.
539-593.

Meites, J. 1962. Pharmacological control of prolactin
secretion and lactation. In; Pharmacological Control
of Release of Hormones Including Antidiabetic Drugs,
Vol. I. R. Guillemin (ed.). Pergamon Press, London.
151—181.

Meites, J. 1963. Neurohumoral and pharmacological control
of prolactin secretion. In: Scientific Group on the
Physiology of Lactation. WOrld Health Organization,
Geneva.

Meites, J. 1966. Control of mammary growth and lactation.
In: Neuroendocrinology, Vol. I. L. Martini and W. F.
Ganong (eds.). Academic Press, New York. 669-707.

Meites, J. (in press). The relation of estrogen and pro-
lactin to mammary tumorigenesis in the rat. In:
Breast Cancer Workshop: Estrogen, Target Tissue and
Neoplasia. T. Dao (ed.). University of Chicago Press,
Chicago.

Meites, J., and T. F. Hopkins. 1961. Mechanisms of oxytocin
action in retarding mammary involution: study in hypo—
physectomized rats. J. Endocrinol. 22: 207-213.

Meites, J., C. S. Nicoll, and P. K. Talwalker. 1963. The
central nervous system and the secretion and release of
prolactin. In: Advances in Neuroendocrinology. A. V.
Nalbandov (ed.). University of Illinois Press, Urbana.
238—277.

Meites, J., and C. L. Kragt. 1964. Effects of a pituitary
homotransplant and thyroxine on body and mammary
growth in immature hypophysectomized rats. Endocrinology
75: 575-570.

Meites, J., and C. S. Nicoll. 1966. Adenohypophysis: Pro—
lactin. Ann. Rev. Physiol. 28: 57-88.

Meites, J., E. Cassell, and J. Clark (in press). Estrogen
inhibition of mammary tumor growth in rats; counter-
action by prolactin. .

58

Mizuno, H., P. K. Talwalker, and J. Meites. 1964. Inhibi-
tion of mammary secretion in rats by iproniazid. Proc.
Soc. Exp. Biol. Med. 115: 604-607.

Muckter, H. 1966. Thalidomide and Tumor. In: Antimicrob-
ial Agents and Chemotherapy. Gladys L. Hobby (ed.).
Ann Arbor, Michigan: American Society of Microbiology.

Muckter, H., E. Frankus, E. More, W. E. Kollmer, and M.
Stoemmler. 1967. Experimentelle Untersuchungen mit
cyclischen imiden bei Dimethylbenzanthracen—tumoren der
Sprague-Dawley ratte. Zeitshrift fur Krebsforschung 69:
60-69.

Muckter, H., E. Frankus, and E. More. 1969. Experimental
therapeutic investigations with l-(morpholinomethy1)-
4-phthalimido-piperidindione-2,6 on Dimethylbenzan-
thracene—induced tumors of SpragueLDawley rats. Cancer
Res. 29: 1212-1217.

Muscholl, E. 1961. Effect of cocaine and related drugs on
the uptake of noradrenaline by heart and spleen. Br.
J. Pharmac. Chemother. 16: 352-359.

Nagasawa, H., C. L. Chen, and J. Meites. 1969. Effects of
estrogen implant in median eminence on serum and pitui-
tary prolactin levels in the rat. Proc. Soc. Exp. Biol.
Med. 132: 859-861.

Nagasawa, H., and J. Meites. 1970. Suppression by ergo-
cornine and iproniazid of carcinogen-induced mammary
tumors in rats; Effects on serum and pituitary prolactin
levels. Proc. Soc. Exp. Biol. Med. 135: 469-472.

Nair, R. M. G., A. J. Kastin, and A. V. Schally. 1971.
Isolation and structure of hypothalamic MSH Release-
Inhibiting Hormone. Biochem. Biophys. Res. Comm.
43: 1376—1381.

Nandi, S., and H. A. Bern. 1961. The hormones responsible
for lactogenesis in BALB/c Crgl mice. Gen. Comp.
Endocrinol. 1: 195-210.

Nelson, W. 0., R. Gaunt, and M. Schweizer. 1943. Effects
of adrenal cortical compounds on lactation. Endo-
crinology 33: 325-332.

Nicoll, C. S., and J. Meites. 1962. Estrogen stimulation
of prolactin production by rat adenohypophysis i3 Vitro.
EndocrinolOgy 70: 272-277.

Nicoll, C. S., and J. Meites. 1963. Prolactin secretion in
Vitro: effects of thyroid hormones and insulin. Endo-
crinology 72: 544-551.

59

Nicoll, C. S., and H. A. Bern. '1964. Prolactin and the
pituitary glands of fishes. Gen. Comp. Endocrinol.
4: 457-471.

Nicoll, C. S., and J. Meites. 1964. Prolactin secretion
in Vitro: effects of gonadal and adrenal cortical
steroids. Proc. Soc. Exp. Biol. Med. 117: 579-583.

Nikitovitch-Winer, M. B., and J. W. Everett. 1958. Com-
parative study of luteotrOpin secretion by hypophysial
autotransplants in the rat. Effects of site and
stages of the estrous cycle. Endocrinology 62: 522-532.

Niswender, G. D., C. L. Chen, A. R. Midgley, Jr., J. Meites,
and S. Ellis. 1969. Radioimmunoassay for rat prolactin.
Proc. Soc. Exp. Biol. Med. 130: 793-797.

Pasteels, J. L., H. Brauman, and J. Brauman. 1963. Etude
comparie de la secretion d'hormone somatotrope par
l'hypophyse humaine ig Vitro, et de son activite
lactogenique. Compt. Rend. Soc. Biol. 256: 2031-2033.

Pearson, O. H., O. Llerena, L. Llerena, A. Malina and T.
Butler. 1969. Prolactin-dependent rat mammary cancer:
A model for man? Trans. Ass. Amer. Physicians 82:
225-238.

Poulson, E., M. Botros, and J. M. Robson. 1960. The effects
of 5—HT and iproniazid on pregnancy. J. Endocrinol.
20: X1.

Ramirez, V. D., and S. M. McCann. 1964. Induction of pro-
lactin secretion by implants of estrogen into the
hypothalamo-hypophysial region of female rats. Endo-
crinology 75: 206-214.

Ratner, A., and J. Meites. 1962. Effect of estrogen
administration 12 vivo on prolactin release in Vitro.
Proc. Soc. Exp. Biol. Med. 112:12-15.

Ratner, A., and J. Meites. 1964. Depletion of prolactin
inhibiting activity of rat hypothalamus by estradiol
or suckling stimulus. Endocrinology 75: 377-382.

Ratner, A., P. K. Talwalker, and J. Meites. 1965. Effect
of reserpine on prolactin-inhibiting activity of rat
hypothalamus. Endocrinology 77: 315-319.

Rivera, E. M. 1964. Interchangeability of adrenocortical
hormones in initiating mammary secretion in vitro.
Proc. Soc. Exp. Biol. Med. 116: 568-572.

6O

Rothchild, I. 1965. Interrelations between progesterone
and the ovary, pituitary, and central nervous system
in the control of ovulation and the regulation of
progesterone secretion. 13: Vitamins and Hormones,
Vol. 23. R. S. Harris, I. G. Wool, and J. A. Loraine
(eds.). Academic Press, New York. 209-327.

Sar, M., and J. Meites. 1967. Changes in pituitary pro-
lactin release and hypothalamic PIF content during
the estrous cycle of rats. Proc. Soc. Exp. Biol. Med.
125: 1018-1021.

Setnikar, I., W. Murmann, and M. J. Magistretti. 1960. The
retardation of sexual development in female rats due to
iproniazid treatment. Endocrinology 67: 511-520.

Shelesnyak, M. C. 1954. Ergotoxine inhibition of deciduoma
formation and its reversal by progesterone. Amer.
J. Physiol. 179: 301-304.

Shelesnyak, M. C. 1955. Disturbance of hormone balance
in the female rat by a single injection of ergotoxine
ethanesulphonate. Amer. J. Physiol. 180: 47-49.

Shelesnyak, M. C. 1958. Maintenance of gestation in
ergotoxine-treated pregnant rats by exogenous prolactin.
Acta Endocrinol. 27: 99-109.

Sokal, R. R., and F. J. Rohlf. "1969. Biometry. W. H.
Freeman and Co., San Francisco. 218-230.

Somers, G. F 1960. Pharmacological prOperties of thalid-
omide (o-phthalimido glutarimide), a new sedative
hypnotic drug. Br. J. Pharmac. Chemother. 15:.

111-116.

Sterental, A., J. M. Dominquez, C. Weissmann, and O. H.
Pearson. 1964. Pituitary role in the estrogen de-
pendency of experimental mammary cancer. Cancer
Research 23: 481-485.

Talwalker, P. K., and J. Meites. 1961. Mammary lubulo-
alveolar growth induced by anterior pituitary hormones
in adreno-ovariectomized and adreno-ovariectomized-
hypophysectomized rats. Proc. Soc. Exp. Biol. Med.
107: 880-883.

Talwalker, P. K., C. S. Nicoll, and J. Meites. 1961. Induc-
tion of Mammary secretion in pregnant rats and rabbits
by hydrocortisone acetate.‘ Endocrinology 69: 802-808.

61

Talwalker, P. K., A. Ratner, and J. Meites. 1963. lg Vitro
inhibition of pituitary prolactin synthesis and release
of hypothalamic extract. Amer. J. Physiol. 205: 213-
218.

Talwalker, P. K., J. Meites, and H. Mizuno. 1964. Mammary
tumor induction by estrogen or anterior pituitary
hormones in ovariectomized rats given 7,12-dimethy1-
1,2-benzanthracene. Proc. Soc. Exp. Biol. Med. 116:
531-534.

Thoenen, H., A. Hurlimann, and W. Haefely. 1964. The effect
of sympathetic nerve stimulation on volume, vascular
resistance and norepinephrine output in the isolated
perfused spleen of the cat and its modification by
cocaine. J. Pharmac. Exp. Ther. 143: 57-63.

Turner, C. W. 1939. The mammary glands. ;_: Sex and In-
ternal Secretions, 2nd ed. E. Allen (ed.). Williams
and Wilkins, Baltimore, Maryland. 740.

Van Maanen, J. H., and P. G. Smelik. 1968. Induction of
pseudopregnancy in rats following local depletion of
monoamines in the median eminence of the hypothalamus.
Neuroendocrinology 3: 177-186.

Wakabayashi, I., and A. Arimura. 1971. Influence of stress
and nembutal on serum prolactin levels in the rat.
Fed. Proc. 30: 254.

Welsch, C. W., J. A. Clemens, and J. Meites. 1968. Effects
of multiple pituitary homografts or progesterone on
7,12-dimethy1benzanthracene-induced mammary tumors in
rats. J. Nat. Cancer. Inst. 41: 465—471.

Whitby, L. G., G. Hertting, and J. Axelrod. 1960. Effect
of cocaine on the disposition of noradrenaline
labelled with tritium. Nature 187: 604-605.

Wiest, W. G., W. R. Kidwell, and K. Balogh, Jr. 1968.
Progesterone catabolism in the rat ovary: a regulatory
mechanism for progestational potency during pregnancy.
Endocrinology 82: 844-859.

Wuttke, W., and J. Meites. 1970. Effects of ether and
pentobarbital on serum prolactin and LH levels in
proestrous rats. ProcI Soc. Exp. Biol. Med. 135:
648-652.

Wuttke, W., and J. Meites. 1971. Luteolytic role of pro-
lactin during the estrous cycle of the rat. Proc.
Soc. Exp. Biol. Med. 137: 988-991.

62

Wuttke, W., E. Cassell, and J. Meites. 1971. Effects of
ergocornine on serum prolactin and LH, and on hypo-
thalamic content of PIF and LRF. Endocrinology 88:
737-741.

Wuttke, W., M. Gelato, and J. Meites (in press). Effects
of pentobarbital on prolactin release.

Zeilmaker, G. H., and R. A. Carlsen. 1962. Experimental
studies on the effect of ergocornine methane-sulphonate
on the luteotrophic function of the rat pituitary
gland. Acta Endocrinol. 44: 321-335.

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