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ABSTRACT

I. LUTEOLYTIC ACTION OF PROLACTIN IN THE MOUSE
II. CHOLINERGIC INHIBITION OF PROLACTIN RELEASE

By
Lindsey Grandison

l. The proestrous surge in serum prolactin was blocked
with ergocornine for one or three cycles in Swiss Webster mice.
Blockade of prolactin release caused a retention of corpora lutea
from earlier cycle(s). Concurrent administration of prolactin and
ergocornine left the number of corpora lutea unchanged since no
differences were noted between this treatment group and controls.
These results indicate that prolactin exerts a luteolytic action
on old corpora lutea during the estrous cycle of the mouse.

2. The effects of acetylcholine injected into the lateral
ventricle of proestrous female rats were observed on serum prolactin.
A 50 pg dose of acetylcholine suppressed prolactin release by 15
and 30 minutes after injection. By 60 minutes after injection the
inhibitory response, although still present, was not significant.
These observations suggest that cholinergic activity in the hypo-

thalamus suppresses release of prolactin.

Lindsey Grandison

3. The effect of a systemic injection of pilocarpine, a
cholinomimetic drug, was noted on prolactin secretion in female and
male rats. In proestrous female rats, pilocarpine at 9 mg/kg supp-
ressed prolactin release by l5 and 30 minutes after injection. At
the end of one hour no depression was observed.

Three doses of pilocarpine were injected into three groups
of male rats. The lowest dose, 5 mg/kg, suppressed prolactin secre-
tion whereas the higher doses had no effect. Higher doses of this
drug may induce stress, a potent stimulator of prolactin release.

4. The effects of several doses of physostigmine, an ace—
tylcholine esterase inhibitor, were noted on prolactin secretion by
30, 60 and 90 minutes after injection. The 0.5 mg/kg dose suppressed
prolactin secretion by 90 minutes, but not by 30 or 60 minutes after
injection. Stress probably was responsible for inducing release of
prolactin by 30 and 60 minutes with this high dose of physostigmine.
These findings indicate that potentiation of cholinergic activity by
physostigmine results in decreased release of prolactin, and supports
the view that a cholinergic system in the hypothalamus helps regulate

prolactin secretion.

I. LUTEOLYTIC ACTION OF PROLACTIN IN THE MOUSE
II. CHOLINERGIC INHIBITION OF PROLACTIN RELEASE

By

Lindseyxérandison

A THESIS

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

MASTER OF SCIENCE
Department of Physiology

1973

Dedicated

to my

Mother and Father

ii

ACKNOWLEDGMENTS

The author wishes to acknowledge with appreciation the
guidance and direction given by Dr. Joseph Meites during the
completion of this program of study. Also the other members of
my guidance committee, Dr. N. Collings, Dr. G. Reigh and Dr. C.
Welsch deserve thanks for their consideration and assistance

during the preparation of this manuscript.

TABLE OF CONTENTS

INTRODUCTION ........................

LITERATURE REVIEW ......................

Luteolysis in Rats and Mice .............. .
Regression of Corpora Lutea During the Estrous Cycle
Regression of Corpora Lutea After Hypophysectomy .
Ergocornine: Effects on Prolactin Secretion .....
The Luteolytic Action of Prolactin During the
Estrous Cycle of the Rat ....... . .......
Luteolysis in the Mouse .......... . .....

Biogenic Amines and Control of Gonadotropin and
Prolactin Secretion ...................
Introduction ................ . . . . .
Relation of Catecholamines to Gonadotropin
and Prolactin Secretion ................
Anatomy of Catecholaminergic Innervation of
the Hypothalamus .................
Effects of Catecholamines on Gonadotropin
and Prolactin Secretion .............
Effects of Adrenergic Drugs on Gonadotropin
and Prolactin Secretion .............
Changes in Content and Turnover of Catecholamines
Associated with Hormone Secretion ........
Relation of Serotonin to Gonadotropin and
Prolactin Secretion .................
Anatomy of Serotonergic Innervation of
the Hypothalamus .................
Effects of Serotonin and Serotonin Precursors
on Gonadotropin and Prolactin Secretion .....
Effects of Serotonergic Drugs on Gonadotropin
and Prolactin Secretion .............
Relation of Acetylcholine to Gonadotropin and
Prolactin Secretion . . ............. .
Anatomy of Cholinergic Innervation of the
Hypothalamus . . ................
Changes in Enzyme Activity Associated With
Various Reproductive States ...........
Effects of Cholinergic Drugs on Gonadotropin
and Prolactin Secretion . . . . . . . . . . . . .

iv

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12
14
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18
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20
21

Page

MATERIALS AND METHODS ....... . . . . , . . . . . . . . 23
Animals ......................... 23
Histological Examination of Ovaries ........... 23
Radioimmunoassay of Prolactin .............. 23
Cannulation of the Lateral Ventricle of Rats ....... 24
Statistical Analysis ................... 25

EXPERIMENTAL ............. . .......... 27
Luteolytic Action of Prolactin During the Estrous
Cycle of the Mouse .................... 27

Effects of Ergocornine Treatment ........... 27
Objective ..................... 27
Materials and Methods ......... . ..... 27
Results ...................... 28
Conclusions .................... 30

Effects of Cholinergic Drugs on Serum Prolactin ..... 3l

Effects of Intraventricular Injection of
Acetylcholine on Serum Prolactin Levels

of Female Rats .................... 3l
Objective . . . . . . . . . . . . . . . . . . . . . 3l
Materials and Methods ............... 31
Results . . . . . . . . . . . . . . . . . . . . . . 31
Conclusions .................... 32

Effects of Pilocarpine on Serum Prolactin

Levels of Male and Female Rats ........ .. . . . 34
Objective ........ , ........ . . . . 34
Materials and Methods ............... 34
Results . . . . .................. 34
Conclusions .................... 35

Effects of Physostigmine on Serum Prolactin

Levels in Male Rats .................. 36
Objective ..................... 36
Materials and Methods ............... 38
Results ........ . ............. 38
Conclusions .................... 38

DISCUSSION ......................... 40
LIST OF REFERENCES ..................... 44

INTRODUCTION

Early investigation into the functions of prolactin
revealed its involvement in the initiation and maintenance of milk
secretion and in the formation and function of the corpora lutea.
With the development of a radioimmunoassay (RIA) for prolactin,
these basic observations were confirmed. Moreover, the greater
sensitivity and specificity of this technique allowed elucidation
of the more subtle functions of prolactin. For example, the recent
observation of a serum prolactin surge during proestrus in the rat
was determined by RIA (Niswender £5.2139 l969). Thereafter Wuttke
et_gl. (197l) inhibited prolactin secretion with ergocornine and
were thus able to show a luteolytic action of prolactin during the
estrous cycle of the rat.

Similarly the understanding of hormone regulation has been
extended through the use of the RIA. Evidence is accumulating that
serotonergic neurons induce the release of prolactin. Thus the
tonic inhibition of prolactin release provided by the catecholaminergic
neurons is countered by a serotonergic stimulation.

These advances in understanding are refinements of existino
concepts. Yet further experimentation is required to substantiate
recent advances and to provide details of the mechanisms regulating
prolactin secretion. Therefore it was considered appropriate to

determine whether prolactin exerted a luteolytic action in a

different Species, the mouse, In addition the recent observation of
a serotonergic stimulus for prolactin release suggested that other
neurotransmitters in the hypothalamus might influence prolactin
secretion. Therefore it was of interest to examine the possible

effects of the cholinergic system on prolactin secretion.

LITERATURE REVIEW

Luteolysis in Ratsand Mice

 

Regression of Corpora Lutea
During the Estrous Cycle

Throughout the reproductive lifespan of laboratory animals
such as the rat and the mouse, spontaneous ovulation is an event in
anticipation of pregnancy. At each ovulation a set of processes re-
quired for pregnancy is begun. However, if mating does not occur,
structures such as corpora lutea cease functioning prematurely. Dur—
ing the estrous cycle of a rat, corpora lutea (CL) develop shortly
after ovulation. In early diestrus they attain their maximal size
(Boling, 1942). Progesterone secretion is maximal on day two of the
cycle. Thereafter secretion of progesterone declines and the less
active metabolite, ZO—a-hydroxypre-4en—3-one (20-OH-P), is secreted
(Hashimoto and Weist, 1969). However, the size of the CL is maintained
until metestrus of the next cycle (Deane, 1952). Between the second
metestrus after formation and the following day, diestrus, a rapid
regression occurs. The remaining structure persists for 8 to 9 days
since at least three generations of CL are normally observed in rats
(Long and Evans, 1922) and mice (Allen, 1922). Although not as well
investigated, the phenomena in the mouse are similar. However, maximal
CL size is not reached until estrus of the second cycle. As in the

rat, CL regress with age. Fat accumulation disappears after the

second metestrus in contrast to the rat where fat accumulation is an
indication of aged CL (Greenwald and Rothchild, 1968).

From the above description luteolysis or regression of the
CL can be seen to include two phases. Malven (1969) has distinguished
between functional luteolysis or cessation of progesterone secretion
and structural luteolysis or morphorological regression. This dis-
tinction is made since these events occur at different times. During
the estrous cycle, pseudopregnancy, pregnancy, and lactation, secre-
tory function ends before structural regression. However, CL can
remain intact for a prolonged time even though secretion has ended.
Such a condition is encountered in the hypophysectomized rat. The
possibility exists that different factors are involved in structural
and functional luteolysis.
Regression of Corpora Lutea
After'Hypophysectomy

The hypophysectomized animal has been used in determining the
anterior pituitary hormones responsible for a number of functions,
Early research demonstrated that prolactin administration immediately
after hypophysectomy was capable of sustaining progesterone secretion
from CL in the rat (Astwood, 1941) and the mouse (Robson, 1971). With-
out exogenous prolactin, the CL of hypophysectomized rats ceased
secreting progesterone within 24 hours. However, in the rat the CL
structure remains for up to 9 months (Smith, 1930) when hypophysectomy
is done during estrus or diestrus. On the other hand, if hypophys-
ectomy is done during proestrus, pseudopregnancy, or if a grafted

pituitary is removed from a hypophysectomized rat with a functioning

set of CL, the CL regress immediately (Greenwald and Rothchild, 1968).
In the mouse connective tissue invasion occurs 12 days after hypo-
physectomy.

The persisting CL of hypophysectomy offer an excellent model
for elucidating the pituitary factors involved in structural luteo- .
lysis. Malven and Sawyer (1966) postponed prolactin injections for
80 hours and found that prolactin caused regression of CL in such
hypophysectomized rats. Also, Piacsek and Meites (1967) found pitui-
tary transplants after hypophysectomy caused luteolysis. LH was not
luteolytic under these conditions and LH was unable to synergize with
moderate doses of prolactin to induce luteolysis (Malven, 1969).
Although a luteolytic activity of prolactin had been clearly demon-
strated, its physiological importance remained undetermined. However,
the early observations of Long and Evans (1922) implicated a physio-
logical condition during which prolactin was luteolytic. In lactating
rats two generations of CL are present, one resulting-from pregnancy
and another resulting from the parturition-induced ovulation.

Normally the CL of pregnancy regress. If the pups are removed and
lactation is prevented, the CL of pregnancy regress much more slowly.
Presumably the suckling induced prolactin surges cause the regression
of the non-functional CL of pregnancy.

After it became well documented that prolactin rose during
proestrus (Amenomori et_al,, 1970; Gay et_al,, 1970) the physiological
significance of this surge was questioned. As a consequence of the
luteolytic action of prolactin in the hypophysectomized rat, it was

considered possible that prolactin might be involved in luteolysis

during the estrous cycle. Before such an investigation could be
undertaken, a means of selectively inhibiting prolactin was needed.
With the determination that ergot alkaloids were effective at low
doses in preventing the release of prolactin (Nagasawa and Meites,
1970), a pharmacological tool was provided.

Ergocornine: Effects on
Prolactin Secretion

 

Ergocornine and other ergot alkaloids are produced by a
fungus which grows on rye and other grains. The ergot alkaloids
were the first alpha-adrenergic blockers discovered, but they also
have a number of other effects. In the early part of this century,
they were used as oxytocics until their untoward side effects were
determined. This action on the uterUs is an example of the direct
stimulation ergots have on smooth muscle, and it is the strongest
peripheral side effect. Its activity as a weak agonist (and there-
fore an occupant of adrenergic receptors, and hence, blocking agent)
is suggested to partially explain smooth muscle stimulation. It is
also an antagonist of serotonin. The central effects cause medullary
depression as evidenced by decreased vasomotor and respiratory
activity, and cause inhibition of hypothalamic temperature regulation.
However, the central action is not believed to be due to any catecho-
laminergic effect. Current therapy utilizes ergot alkaloids in treat-
ment of migraine headaches. Its vasoconstrictive activity is
responsible for relief of this condition.

In addition a number of endocrine effects have been observed.

Ergotoxine, a mixture of ergot alkaloids, inhibited deciduoma formation

(Shelesnyak, 1954) and caused termination of pseudopregnancy and
pregnancy when administered early in these stages (Shelesnyak, 1955).
Injections of prolactin together with ergotoxine were able to over-
come the effects of ergotoxine on pregnancy. -During lactation in the
rat, ergocornine inhibited deciduoma formation and milk production
(Zeilmaker and Carlsen, 1962). These effects were accompanied by
morphological changes in the CL of these animals. These observations
imply that ergocornine disrupts prolactin secretion since pseudo-
pregnancy, pregnancy, lactation and CL function in the rat are
dependent on prolactin. Direct evidence for ergocornine inhibition
of prolactin secretion was provided by Nagasawa and Meites (1970).
Ergocornine was injected for 15 days into rats bearing DMBA induced
mammary tumors. The rats injected with ergocornine had lower serum
and pituitary prolactin than rats injected with the control vehicle.
A single systemic injection has been shown to inhibit the proestrous
rise of prolactin in the serum (Wuttke e_:c___a_1_1_ ., 1971). The site of
action is in part on the median eminence of the hypothalamus since
implants there were effective in increasing PIF and reducing serum
prolactin levels (Wuttke et_al,, 1971). A direct inhibitory effect

on the pituitary also was observed ig_vitro (Lu et a1., 1971).

Luteolytic Action of Prolactin During
the Estrous Cycle of the Rat

The use of ergot alkaloids provided the first evidence that
prolactin has a luteolytic role during the estrous cycle of the rat
(Wuttke and Meites, 1971; Billeter and Fluckiger, 1971). When the

proestrous rise in prolactin is inhibited by ergocornine, the ovaries

retain the old CL and increase in weight as a result. Prolactin
injections together with the ergot alkaloids prevent CL accumulation
and the gain in ovarian weight. These observations are strong evidence
that prolactin during the estrous cycle has the ability to cause re—

gression of the old non-functional CL in the rat.

Luteolysis in the Mouse

 

Considerably less is known about luteolysis in the mouse than
in the rat, and observations in the rat do not always apply to the
mouse. For example, the CL of rats are considered to be moderately
affected by uterine influences. Hysterectomy, ligation or cutting of
the oviduct and utero-ovarian vessels extend pseudopregnancy (Hilliard,
1973). Evidence is accumulating to implicate prostaglandins as the
uterine agents responsible for luteolysis of active CL under a number
of circumstances in rats and other species (Pharris et_al,, 1972). So
far attempts to show a uterine influence on the CL of mice has failed
(Caldwell, 1969; Moor, 1968 a, 1968 b; and Dewar, 1973). These observa-
tions on the mouse imply that non-uterine factors are more important in
the mouse. However, these agents have not been well defined.

Biogenic Amines and Control of Gonadtropin
and Prolactin Secretion

 

 

Introduction

 

The control of anterior pituitary function has been intensively
investigated by endocrinologists. Unlike the posterior pituitary, no
direct innervation of the anterior pituitary exists. An important

advance was made by G. W. Harris (1955) when he showed that vascular

connections between the hypothalamus and anterior pituitary are
necessary for normal anterior pituitary function. His observation
indicated that factors from the hypothalamus must travel through the
portal system to cause the release of anterior pituitary hormones.
Further investigation has resulted in the isolation, identification,
and synthesis of three hypothalamic factors: luteinizing hormone
releasing factor (LRF) (Matsuo et_al,, 1971), thyrotropic hormone
releasing factor (TRF) (Boler et_al,, 1969), and somatostatin (SRIF)
(Brazeau et_al,, 1973). Studies on hypothalamic extracts indicate
that other factors exist in the hypothalamus.

However, these hypothalamic factors are just one system in
the control of anterior pituitary secretion. Even before the re-
leasing factors were isolated, Markee et_al, (1952) provided evidence
of a neutral influence on hormone secretion. These investigators
were able to distinguish a critical period on the afternoon of
proestrus. During this time injection of drugs such as dibenamine
blocked ovulation. Since dibenamine is an adrenergic blocking agent,
these results suggested that a catecholaminergic neuron is triggering
the release of LH. Using neuroactive compounds, others have been
able to implicate neural regulation of the other anterior pituitary

hormones as well.

Relation of Catecholamines to Gonadotropin
and Prolactin Secretion

 

 

Anatomy of Catecholaminergic Innervation
of the Hypothalamus

Chemical analysis provided the first evidence that catecho-

lamines existed in the hypothalamus (Vogt, 1954). Subsequent efforts

10

have shown that both dopamine and norepinephrine are present (Rinne
and Sonninen, 1968).

With the development of histochemical flourescence, the site
of some hypothalamic catecholaminergic tracts was found to be the
ventral mesencephalon, the pons and medulla oblongata (Ungerstedt,
1971). These fibers enter the hypothalamus by the medial forebrain
bundle and innervate the dorsal medial nucleus, periventricular
nucleus, the area ventral to the fornix, the arcuate nucleus and the
internal layer of the median eminence, the retrochiasmatic area, the
paraventricular nucleus, the supraoptic nucleus and the preoptic area.
That these areas are important for hormone regulation has been shown
by lesion and stimulation studies. The preoptic area is important in
the cyclic release of LH (Halasz, 1969). The whole medial basal area
of the hypothalamus is essential for tonic release of anterior pitui-
tary hormones (Halasz, 1969).

In addition there is a dopaminergic pathway extending from
the arcuate nucleus and the ventral portion of the periventricular
nucleus to the external and internal zones of the median eminence
(Fuxe, 1964; Lichtensteiger and Langermann, 1966). The terminals
of these fibers end on cells bordering the primary plexus of the
hypothalamic-pituitary portal system. Fuxe has proposed that these
dopaminergic neurons control the secretion of releasing factors into
the portal system connecting the hypothalamus with the pituitary.
Other investigators have characterized the remaining tracts in the

ventral hypothalamus (Bjorklund, 1968). These tracts include nore—

ll

pinephrine containing neurons extending from above the medic-basal
hypothalamus to the external and internal layers of the median
eminence. Two other tracts have been observed, one reaching the
median eminence, and the other extending only as far as the arcuate
nucleus. Although the transmitter in these last two tracts is a
catecholamine, its identity has not been established.
Effects of Catecholamines on Gonadotropin
and Prolactin Secretion

Direct evidence of catecholaminergic regulation of hormone
release has been provided by studies correlating catecholamine
administration with hormone secretion. Since neither dopamine nor
norepinephrine cross the blood brain barrier, these agents must be
given by a central route. When dopamine is injected into the third
ventricle of estrogen primed, hypophysectomized female rats, systemic
concentrations of LRF and FSHRF increase (Schneider and McCann,
1970a). Likewise collection of blood from the portal system of
dopamine-injected animals contained higher levels of LRF than blood
from animals injected with the control vehicle (Kamberi gt_gl,,
1969). Also administration of dopamine into the third ventricle had
been shown to increase serum LH and FSH and decrease prolactin in
intact female and male rats (Schneider and McCann, 1970b; Kamberi
et_al,, 1970, 1971). Implantation of catecholamines into the median
eminence of the hypothalamus has produced results that do not agree
with the above observations, although direct hormone measurements
were not taken. Instead vaginal smears and ovarian weight were used

to infer long term changes in gonadotropin release in response to a

12

single implantation (Kobayashi and Matsui, l969).

Levo-dopa (l-dopa) is the immediate precursor of dopamine
and easily penetrates the blood brain barrier. Administration of
this compound reduces prolactin secretion from the anterior pituitary
and increases the hypothalamic stores of prolactin release inhibiting

factor (PIF) (Lu and Meites, 1972). In vitro incubation of dopamine

 

with hypothalamic tissue and anterior pituitary causes an increase in
release of LH (Schneider and McCann, 1969).
Effects of Adrenergic Drugs on Gonadotropin
and Prolactin Secretion
The use of adrenergic drugs provided the first evidence for
catecholaminergic regulation of anterior pituitary secretion. Subse-
quently it was shown that drugs that interfere with catecholamine
synthesis or catabolism, alter gonadotropin and prolactin secretion.
Alpha-methyl-para-tyrosine and a1pha-methyl-meta-tyrosine are effec-
tive in inhibiting catecholamine synthesis through substrate competi-
tion. These drugs are also effective in raising serum prolactin
levels (Lu gt_al,, 1970). Castration hypersecretion of FSH and LH in
parabiotic rats was prevented with a1pha-methyl-para-tyrosine
(Donoso and Santolaya, 1969). Ovulation in immature rats injected
with PMS has been blocked by this inhibitor (Lippman et;al,, 1967).
The false transmitter, methyl norepinephrine, is synthesized
from methyldopa and it has little intrinsic activity at adrenergic
receptors. Administration of methyldopa effectively increases serum
prolactin levels (Lu gt_al,, 1970). Also LH secretion was lower in

PMS treated immature rats given methyldopa (Coppola, 1971).

l3

Reserpine depletes the adrenergic neuron of its catecholamine
stores. As with the action of other sympatholytic agents, reserpine
inhibits ovulation in PMS-treated immature rats (Hopkins and Pincus,
1963). Reserpine has also been shown to reduce synthesis and release
of LH and FSH in intact and castrated rats (Gronroos et_al., 1965;
Labhsetwar, 1967) and to increase prolactin release in intact rats
(Lu et_al., 1970).

Adrenergic receptor blockers were very prominent in the in-
vestigation of nervous regulation of anterior pituitary secretion.
Dibenamine was found to inhibit ovulation presumably because it
blocked adrenergic stimulation (Markee et_al., 1952). Other
alpha-adrenergic blockers have been found to influence hormone
secretion. Chlorpromazine blocked the PMS induced rise in LH in
immature rats (Zarrow and Brown-Grant, 1964). Since human chlorionic
gonadotropin overcame the Chlorpromazine effect, the action of this
blocker is believed to be central. Prolactin secretion is stimulated
by Chlorpromazine (Lu et_al., 1970) and by the dopaminergic receptor
blockers, haloperidol (Dickerman et_al,, 1972) and pimozide (Meites
and Clemens, 1972).

In contrast to the above agents monoamine oxidase inhibitors
such as pargyline and iproniazid increase the neuronal concentration
of catecholamines by slowing their catabolism. Predictably these
drugs decrease prolactin release (Donoso em” 1971).

The effect of the above drugs which penetrate the blood-brain
barrier and of the catecholamines is believed to be central. Many

peripheral acting drugs do not have the ability to interfere with

14

hormone secretion. Several laboratories (Schneider and McCann, 1969;
Kamberi gt;al,, 1970) have found dopamine to be ineffective for in-
ducing LH release from the pituitary directly. However, dopamine,
norepinephrine, and epinephrine can act directly on the pituitary to
inhibit the release of prolactin (Jacobs et_al., 1968; MacLeod, 1969;
and Birge gt_al., 1970). As yet no catecholamines have been detected
in the hypothalamo-pituitary portal blood, and until they are found
in the portal blood catecholamines cannot be considered to have an
effect on the pituitary directly under normal psysiological conditions.
Changes in Content and Turnover of
Catecholamines Associated With
Hormone Secretion

The most physiological approach to hormone control involves
correlation of neuronal activity with rates of hormone secretion from
the anterior pituitary. In this regard it was noted that after cast-
ration the anterior hypothalamic content of norepinephrine is
increased (Donoso gt_al., 1967; Stefano et_al,, 1965) and tyrosine
hydroxlyase activity is increased (Beattie gt_al,, 1972). Synthesis of
norepinephrine is also accelerated after castration (Anton-Tay gt_gl,,
1970; Wurtman §t_al,, 1969; and Anton-Tay and Wurtman, 1968). Admin-
istration of estradiol or estradiol plus progesterone to castrated
animals suppresses increased synthesis and content of norepinephrine
(Donoso and Stefano, 1967; Bapna gt_al,, 1971; Beattie et_al,, 1972).
During the estrous cycle, norepinephrine content of the anterior
hypothalamus is minimal during estrus, increases during diestrus and

peaks at proestrus (Stefano and Donoso, 1967). Catecholamine

15

synthesis from exogenous tritiated tyrosine is highest on proestrus

and lowest on diestrus (Zschaeck and Wurtman, 1973). At puberty, when
LH and FSH are required for vaginal opening, hypothalamic catecholamine
content and synthesis rate are increased (Coppola, 1968, 1969).

However, not all investigations agree with the above results.
Fuxe and Hokfelt (1969), using the semiquantitative histochemical
florescence technique, concluded that catecholamines inhibit gonado-
tr0pins and promote prolactin secretion. Fuxe and Hokfelt noted no
change in norepinephrine content after castration or during pregnancy
or lactation. Total catecholamine content was found to increase
during diestrus while dopamine was unchanged. Dopamine was increased
during pregnancy, pseudopregnancy, and lactation but did not change
after castration.

These controversies arise because not all investigators agree
on the most appropriate index of neuronal activity. Also recent
advances in neurochemistry make interpretation of these results diffi-
cult. It has been accepted that the concentration of norepinephrine
is under feedback control so that in norepinephrine containing neurons,
synthesis rate is an appropriate measure of neuronal activity. On the
other hand, Walters and Roth (1973) have recently shown that dopamin-
ergic neurons increase synthesis of dopamine when deprived of presyn-
aptic stimulation. Hopefully further efforts in this area will
resolve the existing contradictions and provide direct evidence linking

catecholaminergic neuronal activity with anterior pituitary secretion.

16

Relation of Serotonin to Gonadotropin
and Prolactin Secretion

Anatomy of Serotonergic Innervation
of the Hypothalamus

Serotonergic innervation of the hypothalamus has not been as
thoroughly explored as the catecholaminergic tracts. It has been
established that the cell bodies of serotonergic neurons are located
on the raphe region of the mesencephalon and project into the hypo-
thalamus via the medial forebrain bundle (Dahlstrom and Fuxe, 1964;
Fuxe, 1965). Serotonergic terminals have been observed in the
suprachiasmatic nucleus (Fuxe, 1965), in the middle of the retro-
chiamatic area, and in the anterior median eminence (Fuxe and
Hokfelt, 1970). Chemical analysis has also shown the presence of high
concentrations of serotonin in the bovine median eminence (Piezzi,
1970). In addition incubation experiments demonstrate the ability
of the median eminence to synthesize serotonin from tryptophan
(Hamon, gt_gl,, 1969).
Effects of Serotonin or Serotonin
Precursors on Gonadotropin and
Prolactin Secretion

Serotonin like the catecholamines, does not readily cross the
blood brain barrier (Douglas, 1971). 7However, when serotonin is given
centrally, it increases prolactin (Ramberifet;al,, 1970, 1971). Im-
plantation of serotonin directly into the median eminence is followed

by a decline in serum LH levels (Fraschini, 1970). Systemic injection

of 5 hydroxytryptophan, a precursor of serotonin that crosses the

17

blood brain barrier, increases serum prolactin (Lu and Meites, 1973)
while it blocks ovulation in PMS treated immature rats (Kordon gt_al,,
1968).
Effects of Serotonergic Drugs on
Gonadotropin and Prolactin
Secretion

Complications arise in interpretation of studies using some
drugs. Reserpine and the other rauwolfian alkaloids interfere with
the binding of both catecholamines and serontonin in the synaptic
vesicle. Since the catecholaminergic and serotonergic system appear
to counteract the actions of each other, the observations after
reserpine injection might result from an effect on one or both systems.
The same problems are encountered with monoamine oxidase inhibitors,
since these compounds interfere with the enzyme metabolizing both
catecholamines and serotonin. Kordon gt_al., (1968) have blocked the
synthesis of either serotonin or catecholamines prior to injection
of a monoamine oxidase inhibitor. They concluded that monoamine
oxidase inhibitors block ovulation by increasing extra-neuronal
serotonin levels. A more specific drug, p-chlorophenylalanine
(p-CPA), which blocks the synthesis of serotonin, increased the
number of eggs ovulated by PMS treated immature rats (Kordon and
Glowinski, 1972). Furthermore the intensity of provoked ovulation
in immature animals is inversely proportional to the concentration of
serotonin in the hypothalamus (Kordon, 1970).

Another indication of serotonin involvement in anterior

pituitary secretion comes from studies on diurnal variations in

18

brain serotonin content and prolactin secretion. Brain serotonin
concentration is believed to be regulated by the blood levels of
tryptophan in the brain (Fernstrom and Wurtman, 1971). It has been
found that there is a daily fluctuation in blood levels of trypto-
phan with a maximum eight hours after lights are turned on and a
minimum four hours after lights are turned off (Wurtman e§_§l,,
1968; Fernstrom and Wurtman, 1971). A similar change in serum
prolactin has been noted with highest values in the afternoon (Koch
gfl;j{L., 1971). Pituitary content of prolactin also varies and reaches
its peak late in the afternoon (Clark and Baker, 1964).

Relation of Acetylcholine to

Gonadotrgpin and Prolactin
Secretion

Anatomy of Cholinergic Innervation
of the Hypothalamus

The cholinergic tracts within the hypothalamus have not been
well described. The techniques available for detectiOn of cholinergic
neurons are not as sensitive or as specific as histochemical floures-
cence. However, some information has been gathered using a variety of
techniques.

In one approach, areas responsive to cholinergic neurons are
revealed by recording from a region while acetylcholine is applied.
Such studies have indicated that the dorsal and caudal regions of the
hypothalamus are facilitated by acetylcholine whereas the rostral and
ventral regions are inhibited (Bloom, gt_§l,, 1963). These results

show the regions effected by acetylcholine but are not direct

19

evidence of cholinergic innervation.

A second approach utilizes a histochemical technique for
locating acetylcholine esterase (AchE). This enzyme splits acetyl-
choline and is the mechanism for terminating cholinergic stimulation
at a synapse. Both cholinergic and cholinoceptive neurons, those
stimulated by acetylcholine, contain this enzyme. These neurons
can be distinguished from one another by the intracellular location
of AchE. Using this approach Shute and Lewis (1969) demonstrated
that the perifornical nucleus and the dorsal-posterior region of the
hypothalamus possess many cholinergic fibers. They noted several
tracts within the hypothalamus. One extends from the lateral preoptic
area to the supraoptic nucleus and then projects to the amygdala.

The supraoptic nucleus was also observed to contain cholinoceptive
neurons. A second pathway originates in the supramammillary region

and leads to the mammillary bodies, specifically the medial and the
lateral mammillary nuclei. Additional tracts ascend from the dorsal-
caudal hypothalamus to the thalamus. There also is a cholinergic
pathway of the reticular system which passes through the hypothalamus.
In addition AchE is found in the arcuate nucleus but in low concentra-
tion. It was not determined whether fibers in this area are cholinergic
or cholinoceptive.

Although lesion and diafferentation studies emphasize the
importance of the anterior and basomedial hypothalamus, lesions in
the thalamo—hypothalamic border and dorsocranial mesencephalon, the
sites of cholinergic neurons, produce increased prolactin release

(Chen et al., 1970; Flerko, 1966; Flerko and Bardos, 1966).

20

Others have found acetylcholine in the median eminence
(Kobayashi et_al;, 1966). Kobayashi and Matsui (1969) noted axonal
endings in the median eminence with small vesicles. These vesicles
resemble synaptic vesicles containing acetylcholine in the periphery
and are distributed similarly to AchE. It was suggested by these
investigators that such axons are cholinergic.

Changes in Enzyme Activity Associated
With Different Reproductive States

Several investigators have monitored acetylcholine esterase
or choline acetyltransferase activity during various reproductive
states in order to determine whether any relationship exists.
Kobayashi gt_al. (1966) measured the activity of choline acetyl-
transferase, the enzyme synthesizing acetylcholine, in the anterior
and posterior hypothalamus. During the estrous cycle, choline acetyl-
transferase activity was lowest in proestrus and estrus in the
anterior hypothalamus while the posterior hypothalamus showed no
change in the activity of this enzyme. After castration choline
acetyltransferase activity increased in the posterior hypothalamus.
When estrogen was given to these castrated animals, the choline
acetyltransferase activity decreased. In prepuberal females, estrogen
caused ovulation and increased choline acetyltransferase activity at
the same time.

Recently Libertun gt_al;_(l973) measured choline acetyl-
transferase and acetylcholine esterase activity in the anterior and

the posterior hypothalamus. Both enzymes showed higher activity

21

during diestrus in the preoptic-suprachiasmatic area while no varia-
tion was seen in the arcuate-mammillary region. Lower enzyme
activity was seen in males than in females. This difference in enzyme
activity between sexes might be a reflection of hypothalamic matura—
tion. After birth, testosterone from the male testes causes the
hypothalamus to induce secretion of gonadotropins and prolactin at a
constant baseline level. In the female the hypothalamus induces a
cyclic pattern of hormone release. The rise in enzyme activity by
25 days of age in males castrated at birth suggests that hypothalamic
maturation might determine cholinergic enzyme activity. However, a
similar rise occurs in the females when castrated early. Consequently
the relationship between enzyme activity and hypothalamic maturation
is not clear.
Effects of Cholinergic Drugs on
Gonadotropin and Prolactin
Secretion

In the early studies on anterior pituitary regulation,
atropine, a cholinergic blocking agent was observed to inhibit ovula-
tion in the rat (Everett et_al,, 1949) and in the rabbit (Sawyer et_
31,, 1949). Synthetic antimuscarinic agents such as methantheline and
pathelon, which act like atropine, also block ovulation (Sawyer, 1963;
Gitsch and Everett, 1958). Recently injections of atropine into the
third ventricle or beneath the skin were observed to inhibit the
proestrous surge of the gonadotropins and prolactin (Kamberi and
Bacleon, 1973; Libertun and McCann, 1973). These effects of atropine

are believed to be central since LRF can overcome the atropine

22

blockade of ovulation (Libertun and McCann, 1973). Under 12.31339.
conditions, atropine inhibited LH secretion from pituitaries
co-incubated with ventral hypothalamic tissue (Kamberi and Bacleon,
1973). In contrast acetylcholine co-incubated with pituitary tissue
and hypothalamic tissue stimulated LH secretion.

Prolactin secretion is influenced by atropine but the type
of effect is dose-dependent. High doses injected systemically induced
lactation (Meites et_al,, 1960) and pseudopregnancy (Gitsch and
Everett, 1958), while low doses inhibited lactation (Jacobson, et_al.,
1950) and pseudopregnancy (Grosvenor and Turner, 1958). Cholinomimetic
agents such as pilocarpine, a direct agonist, or physostigmine, a
acetylcholine esterase inhibitor, induced lactation in rats (Meites
et_al,, 1960). Lactation is dependent on other hormones, principally
adrenal cortical steroids, besides prolactin and is induced by stress
also. Thus lactation is not a specific indicator of prolactin
secretion.

When these cholinomimetic agents are given to ovariectomized,
estrogen primed rats, LH and prolactin decrease and then increase
(Libertun, 1973). Such results are inconsistent with the implications
of atropine administration. However, the use of atropine has some
objections since it has a number of side effects, especially at the
high doses used. Consequently more research is required to establish

the role of cholinergic neurons on pituitary function.

MATERIALS AND METHODS

Animals

Mature 2 to 3 month old virgin female Swiss-Webster mice and
mature virgin female and mature male Sprague-Dawley rats were obtained
from Spartan Research Animals, Inc., Haslett, Michigan. All animals
were housed in a temperature controlled (75:10F.) artificially
illuminated room with 14 hours of light (5:00 a.m. to 7:00 p.m.) and
10 hours of darkness. Wayne Lab Blox pellets (Allied Mills, Chicago,
Illinois) and water were provided gg_lib,

Estrous cycles were determined by taking daily vaginal smears
and only those females showing at least two regular 4 or 5 day cycles

were used for experimentation

Histological Examination of Ovaries
For histological examination of the ovaries from mice, they
were removed, cleaned, rolled dry on a piece of kemi—wipe and weighed
on a torsion balance. The tissues were then fixed in Bouin's fluid
and stained with hematoxylin and eosin. Sectioning was done parallel

to the longest axis at the center of each ovary.

Radioimmunoassay of Prolactin
Serum prolactin concentration was determined by radioimmuno-
assay as described by Niswender et a1. (1969). Serum samples were

diluted with 0.1% gelatin in phospho-buffered saline. At least two

23

24

dilutions of each sample were made and the mean of the dilutions was
used as the sample value. The sample values were expressed in terms
of a standard: National Institute of Arthritis and Metabolic
Diseases-Rat Preparation-1 (NIAMD-RP-l) obtained from the National
Institutes of Health, Bethesda, Maryland. The prolactin used for
iodination was provided by Dr. 5. Ellis of NASA Ames Research Center,

Moffet Field, California. Radioactive iodine, 1‘25

, was supplied by
Amersham/Searle, Chicago, Illinois. The first antibody was collected
from a rabbit and characterized by Niswender gt_gl,(1969). The
second antibody was collected from a sheep kept locally and diluted

as titration indicated.

Cannulation of the Lateral Ventricle of Rats

Lateral ventricle cannulation followed the description of
Verster gt_al, (1971). Female rats weighing 200 to 225 grams were
anesthetized with sodium pentobarbital (45 mg/kg). .After placement
in a Stoelting or Neuman stereotaxic instrument the skin was retracted
and two holes were drilled through the skull with a small hand drill.
The hole receiving the cannula was drilled l millimeter behind and
2 millimeters lateral to bregma. The cannula was made from poly-
ethylene tubing (PE 10) by inserting a wire in the tubing, heating
over a soldering gun and compressing the heated region so that a bulb
formed. The tubing was cut 4 millimeters from the bulb with a l milli-
meter bevel. Five centimeters of tubing were left on the other side
of the bulb and this end was heat sealed. When placed in the hole,

the bulb of the cannula rested on the surface of the skull, and the

25

short end protruded into the lateral ventricle. The second hole was
fitted with an anchor screw. Both were cemented in place with dental
cement. The skin was sewn and the animals were injected with 0.2
milliliter of Longicillin, (Fort Dodge Laboratories, Inc., Fort Dodge,
Iowa) an antibiotic. Vaginal smears were taken after cannulation to

ascertain that cycling continued.

Statistical Analysis

Means and standard errors of the means were calculated. The
significance of difference between a control group and a treated
group was determined by Student's t test. Where numerous treatment
means were compared to a single control mean, Dunnett's multiple range
test was used after an analysis of variance indicated treatment
differences.

The following formulae were used in determining statistically
significant differences:

1. Student's t test:
t value for the Student's t test

=T-Ho

a Y
where T represents the treatment mean
no represents the control mean
67 represents the standard derivation.

Sokal and Rohlf, 1969.

26

2. Dunnett's multiple range test:
d' the difference which must be found between a treatment mean and
the control mean before significance is achieved according to the
Dunnett test.

= t Da/2; k,v\\/2(MS error)
n

 

t Da/Z; k,v - a value obtained from a Dunnett's table
n = number of animals per group
MS error - the error mean square

Dunnett, 1970.

EXPERIMENTAL

Luteolytic Action of Prolactin During
The Estrous Cycle of the Mouse

Effects of Erggcornine Treatment,

Objective

Recent work has shown that the proestrous surge of prolactin
during the estrous cycle of the rat is responsible for luteolysis
of old corpora lutea (Wuttke and Meites, 1971; Billeter and
Fluckiger, 1971). Prolactin also rises on proestrus in the mouse
(Kwa and Verhofstad, 1967) and regression of old corpora lutea occurs.
It was of interest, therefore, to determine in the mouse whether
inhibition of prolactin secretion by the drug ergocornine during
proestrus and estrus could prevent luteolysis of the old corpora
lutea, and whether concurrent administration of prolactin could induce
luteolysis. Previously ergocornine was reported to decrease pituitary

prolactin levels in the mouse (Yanai and Nagasawa, 1970).

Materials and Methods

Twenty mature Swiss-Webster mice were injected intraperiton-
eally with 200 mgs. of ergocornine methanesulfonate base (Sandoz, Ltd.,
Basel, Switzerland) daily, dissolved in 70% ethanol first and then in

0.9% saline to give a final concentration of 4% ethanol. Treatment

27

28

was begun on the afternoon of the diestrous day prior to proestrus
and continued for three days. Ten of these mice received 1 mg. of
prolactin (NIH-P-S-B ovine prolactin, 28 IU/mg) dissolved in 0.9%
saline on the afternoon of proestrus and on the morning of estrus.
In a group of ten contrdl mice, each was injected intraperitoneally
with the saline-ethanol solution during the same period. At the end
of treatment, on the first day of diestrus, the mice were killed and
the ovaries were removed, weighed and prepared for histological
examination.

In addition, 16 mice were injected intraperitoneally daily
with 200 ug of ergoncornine methanesulfonate per mouse beginning on
the afternoon prior to the expected day of proestrus and continuing
for three estrous cycles. During each cycle, 9 of the 16 mice were
injected with 1 mg. of prolactin on the afternoon of proestrus and
on the morning of estrus during each cycle. A control group of 8
mice was injected daily for three cycles with the saline—ethanol
solution. The mice were killed on the first day of diestrus after
the third cycle, and the ovaries were removed, weighed and prepared

as above.

Results

Ergocornine treatment during a single cycle (Table 1) led
to a significant increase in the amount of corpora lutea (9.7 i 0.7)
compared to the number of corpora lutea (6.0 i 0.4) in control mice.
Injections of prolactin on the days of proestrus and estrus in

ergocornine-treated mice resulted in a return of the number of corpora

29

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.cmwe mg» Ho Loggw ugmucmpm H cause

 

 

 

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3O

lutea to about the same number as in control mice. No differences were
apparent in the appearance of the corpora lutea as a result of ergocor;
nine treatment. 0varian weight was slightly reduced in the group given
ergocornine and prolactin compared to the other two groups. None of
the groups differed significantly in body weight.

Ergocornine treatment during three cycles had the same effect
as treatment during one cycle, resulting in a significant increase in
number of corpora lutea compared to those in control mice. Prolactin
administration to ergocornine-treated mice on the days of proestrus
and estrus during each of the three cycles resulted in a return of
corpora lutea to about the same number as in normal mice. No signifi-
cant differences were observed in ovarian or body weights in any of

the three groups of mice.

Conclusion

These results demonstrate that a reduction in prolactin
secretion in cycling mice induced by ergocornine reSults in an increase
in numbers of corpora lutea and that injections of prolactin on the
days of proestrus and estrus returns the number of corpora lutea to
normal. It is apparent, therefore, that prolactin promotes luteolysis
of corpora lutea during the estrous cycle of the mouse as it does in
the rat. Unlike the rat, the accumulation of corpora lutea in the
ovaries in the mouse as a result of ergocornine treatment was not
accompanied by increased ovarian weight. Others also found no increase
in ovarian weight in ergocornine-treated mice (Yanai and Nagasawa,
1970). No obvious differences were observed in histological appearance

of the corpora lutea of the mice treated with ergocornine as compared

31
with the corpora lutea of control mice.
Effects of Cholinergic Drugs on Serum Prolactin

Effects of Intraventricular Injection
of Acetylcholine on Serum Prelactin

Objective

Presence of biogenic amines within the hypothalamus suggests
they may regulate anterior pituitary function. Infusion into the
brain of catecholamines and serotonin has demonstrated that they alter
anterior pituitary secretion (Kamberi gt_al., 1970, 1971). Therefore,
it was of interest to determine whether acetylcholine, which is also
present in the hypothalamus, could influence prolactin release from

the anterior pituitary when given intraventricularly.

Materials and Methods

Mature female rats (200-225 grams) were implanted with poly-
ethylene tubing for lateral intraventricular injection.1 At 11:00 a.m.
on proestrous day the rats were slowly infused with 50 ugs. of acetyl-
choline bromide (K and K Laboratories, Inc., Cainscrew, New York) in
a volume of 8 ul of 0.85% NaCl. Controls were infused with an equal

volume of 0.85% NaCl. Blood was collected under light ether anesthesia

by cardiac puncture 15, 30 and 60 minutes after infusion.

Results
Injection of acetylcholine directly into the lateral

ventricle of the brain produced a 47% decrease in serum prolactin

32

by the end of 15 minutes (Table 2). This reduction in serum prolactin
was also observed at 30 and 60 minutes after injection, although the
60 minute values were not different statistically from the control

values.

Conclusion

Acetylcholine does not cross the blood brain barrier when
given systemically (Koelle, 1970). However, the present results show
that acetylcholine given intraventricularly does cause a rapid de-
pression in serum prolactin. The short duration of this effect is
probably due to its rapid metabolism by the choline esterases. Pre-

vious studies showed no direct effect of acetylcholine on ig_vitro

 

pituitary release of prolactin (Talwalker et al., 1963) or gonado-
tropins (Kamberi and Bacleon, 1973). The effect on prolactin release
may be the result of acetylcholine acting directly on the hypothalamus

to increase PIF release or to decrease release of PRF.

33

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came an» mo Loggm ngmucmpm H names

 

 

m H mm an H mm mm H op AN—V wquoLm wcmpochAHmu<
m H ow n H me am H om Ampv mpogpcou
.cwE om .cwE om .cwE mp mama Ho .02

new HemEHmmgh

 

 

mama apnea; mzogpmmoga oHcH “a; omv muweogm mcwpogopzumo< we
cowpomncH gmpzowgucm>mspcm Lme< Piamiaz<Hz Ho ~s\mc cw cwpom_oga Escmm .N mpnmp

34

Effects of Pilocarpine on Serum
Prolactin Levels of Male and
Female Rats

Objective

Pilocarpine is an agonist of acetylcholine which can pass
through the blood brain barrier. Systemic administration of this
drug would be expected to reproduce cholinergic activity within the
central nervous system. Therefore, this drug was used to test its

effect on prolactin release by the pituitary.

Materials and Methods

Mature male rats were injected with pilocarpine nitrate
(Nutritional Biochemical Corporation, Cleveland, Ohio) intraperiton-
eally at doses of 5, 10 and 18 mg/kg. Female rats were injected with
9 mg/kg of the drug intraperitoneally at noon on the day of proestrus.
Blood was collected by cardiac puncture under light ether anesthesia

15, 30 and 60 minutes after injection.

Results

In male rats pilocarpine at the 5 mg/kg dose significantly
reduced serum prolactin (Table 3). A dose of 10 mg/kg also reduced
serum prolactin values although these differences were not signifi-
cant. The highest dose, 18 mg/kg, apparently had no effect on serum
prolactin. This may be due to the stressful effects of this dose
since diarrhea and piloerection were observed in these rats. When

pilocarpine was administered systemically to female rats at a dose

35

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HmmH H m.H:mu=Hm .ngu m> mPochoo No.ov no

a
A v cm cmuwuwvcw mpg; #0 .oz

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cmmE up: we LOLLm ULMUCMHVI H cams—.1

 

 

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-------- Hfl_vm.H H e.m_ m.m H _.H_ NH H mee Amy ax\me o—
eflmvm.e H o._P e.eAmvm H e.m .eA_vm.N H m.m o_ H mes any me\me m
o.m H _.__ m.e H a.mm m.e H m.e~ HN_ H wee Aev H_eeHeoo
.eee om .eee om .eHe m_ Amv Heme Ho .02

.Hz atom ecu HcmeHmmch

 

 

HHem ape: cw eHeeHHz

mcwncmquHQ wo cowpumhcm Pmmcopwcmamgch LmHe< Fimmioz<Hz mo _s\m: c? :HHUHFoca Ezgmm .m m_nmh

36

of 9 mg/kg, significant reductions in serum prolactin were observed
after 15 and 30 minutes but not by 60 minutes after injection

(Table 4).

Conclusion

These results show that pilocarpine can cause a reduction
in serum prolactin in both male and female rats. A low dose
(5 mg/kg) was most effective in males, whereas 9 mg/kg was effective
in females. A dose of 10 mg/kg did not produce a statistically
significant reduction in serum prolactin in males. The difference
in the number of animals used in the two experiments may be the
explanation for this inconsistency. [The highest dose of pilocarpine
appeared to stress the rats. Since stress is a potent stimulus for
prolactin release, the depressive effect of this dose might have
been overcome by the stress. The results suggest that cholinergic
stimulation produced by pilocarpine reduces release of prolactin
from the anterior pituitary.

Effects of Physostigmine on Serum
Prolactin Levels in Male Rats

Objective

An injection of cholinomimetic drugs can stimulate Chquque.

1‘ H.
1’} 1,4"

ceptive neurons, but they might also have a nonspecific effect. For
this reason another approach was sought to test the effect of chol-
inergic stimulation on prolactin secretion. Physostigmine is a

reversible inhibitor of choline esterase, the enzyme degrading

37

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some wzu mo Loggm ugmucmym H :mmse

 

 

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mcwagmoopwa

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mcwagmooFHa Ho coHHumwcH Pmmcopwcmamguc~ Loue< Fiamioz<Hz Ho _E\m= :H cHHUHFoLm Eaemm .H mpnmp

38

acetylcholine in the synaptic cleft and terminating its action. In-
jection of physostigmine can prolong the effect of acetylcholine
released at a synapse, thus potentiating the effects of cholinergic
activity. Therefore, physostigmine was used to determine the effect .

on prolactin secretion of normal cholinergic activity.

Materials and Methods

Male rats were injected intraperitoneally with physostigmine
sulfate (Merck and Co., Rahweg, New Jersey) in doses of 0.3, 0.4 and
0.5 mg/kg. Blood samples were taken under light ether anesthesia by

cardiac puncture 30, 60 and 90 minutes after injection.

Results

The two lower doses of physostigmine, 0.3 and 0.4 mg/kg,
were effective in suppressing prolactin release during the three time
intervals observed (Table 5). The highest dose, 0.5 mg/kg, was in-
effective at 30 minutes. At 60 minutes a non-significant depression
was noted, but by 90 minutes this dose also caused a depression of

prolactin release.

Conclusion

These results show that physostigmine causes a depression in
serum prolactin. The highest dose may have a stress effect which
stimulates prolactin, thus causing a competition of effects. The
suppression of serum prolactin by physostigmine is probably related
to its potentiation of cholinergic activity. Thus these results suggest

that cholinergic activity can produce a depression in serum prolactin.

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DISCUSSION

A luteolytic role for prolactin has previously been demon-
strated in the rat but not in the mouse. The results reported here
indicate that prolactin also causes regression of old, non-functional
corpora lutes in the mouse. This supports the view that the pro-
estrous surge of prolactin is the physiological mechanism for
accomplishing luteolysis during the estrous cycle of the mouse. A
possible complication is the involvement of LH in luteolysis.
Rothchild (1965) has proposed that LH is the luteolytic agent of the
pituitary in the rat. A recent study by Hausler and Malven (1971)
clearly demonstrates a synergism between prolactin and LH in
luteolysis of functioning corpora lutea in this species. Still
undetermined, however, is the question of the action of LH on
non—functioning corpora lutea. If one can consider structural
regression as a phenomenon separate from cessation of progesterone
secretion, then much of the accumulated data on the luteolytic
action of LH relates to functional luteolysis but not to
non-functional corpora lutea. Malven (1969) found that LH was unable
to induce regression of non-functional corpora lutea and that it was
even unable to synergize with sub-minimal doses of prolactin in in-
ducing regression. Before one can propose that luteolysis during the
estrous cycle is due entirely to prolactin, the involvement of LH

must be excluded.

40

41

Also unresolved is the apparent opposite actions of
prolactin on luteal tissue. It is luteotrophic on newly developed
corpora lutea, while in older corpora lutea it is luteolytic. The
length of the time interval before prolactin is present may explain
the difference in reactivity of the corpora lutea. Thus within 56
hours after hypophysectomy an injection of prolactin is luteotrophic,
but after 80 hours a prolactin injection is luteolytic. Another
possibility is that the corpora lutea may need to be primed by other
hormone(s) in order to grow and secrete progesterone under the
influence of prolactin. Hilliard (1973) suggested that estrogen is
important in determining the reactivity of luteal tissue to pro-
lactin. As evidence, she cited a study by Hixon and Armstrong (1971)
in which prolactin was found to be luteolytic when administered 4
days after destruction of antral follicles by irradiation. If,
however, estradiol was given daily after the radiation treatment,
prolactin was luteotropic. Armstrong (1968) also found that prolactin
does not increase progesterone release in hypophysectomized rats
unless LH is administered 28 hours earlier. The LH in this case
would cause an increase in estrogen synthesis.

During the estrous cycle the old corpora lutea are sub-
jected to estrogen action since estrogen rises late on diestrus day
two and is the stimulus for the proestrous release of LH and pro-
lactin. Either the corpora lutea are incapable of being primed by
estrogen because of age or the corpora lutea do not react soon

enough to the estrogen before prolactin induces regression.

42

Obviously additional research is required to determine the factors
regulating the reactivity of the corpora lutea to prolactin. This
study clearly demonstrates luteolytic action of prolactin during the
estrous cycle of the mouse.

The present work provides evidence for a cholinergic
control of prolactin secretion. Direct infusion of acetylcholine
decreased serum prolactin. Similar results were obtained by
systemic injection of other cholinomimetic agents. However, high
doses of physostigmine or pilocarpine did not change serum prolactin,
since stressful agents generally increase serum prolactin levels.
Although these results agree with the very recent observations of
Libertun (1973), they contradict the implications of studies using
the cholinolytic drug, atropine. This drug blocked ovulation and
decreased prolactin in estrogen-primed ovariectomized animals, thus
implying that cholinergic neurons stimulate prolactin secretion.
Resolution of this controversy necessitates additional work. However,
some comments are appropriate in respect to atropine administration.
The dose of atropine given subcutaneously to inhibit ovulation was
90% of the LD 50 (Cahen and Tvede, 1952). Stress alone is a potent
inhibitor of ovulation and it cannot be overlooked in this case. Also
noteworthy is the antiserotonergic (Jacques, 1956) and anticatechola-
minergic properties of atropine. These latter properties of atropine
could explain its action on ovulation.

The site of cholinergic action induced by the cholino—
mimetic agents is probably the central nervous system as indicated

by intraventricularly administered acetylcholine. Although there

43

are cholinergic neurons in the hypothalamus, these may not be the
ones;eaasipg tNe response to pilocarpine and physostigmine. Cholino-
ceptive neurons outside the hypothalamus but with connections to it,
might be the initial site of action. The effects on LH secretion of
pituitaries ig_yjt£g_co-incubated with hypothalamic tissue and
acetylcholine argue against an extra-hypothalamic site of action
(Kamberi and Bacleon, 1973). It is unlikely that these agents are
directly effecting the pituitary since acetylcholine does not induce
release of LH when incubated with pituitary tissue (Kamberi and
Bacleon, 1973), and early work showed no change in prolactin secretion
when acetylcholine was added to incubated pituitaries (Talwalker
et_al,, 1963).

There remains the more difficult task of relating these
observations to physiological phenomena. It is interesting that
cholinomimetic agents depress prolactin. However, no physiological
role for cholinergic activity on prolactin secretion has yet been
demonstrated. There also is the need to learn the possible re-

lationships between acetylcholine and the important catecholaminergic

and serotonergic systems in the control of prolactin secretion.

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