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THE

Effie
Prolactin an
female rats,
male and ferr
month old LC
light ether.
Samples Were
stress On t}
day of Stre:
meaSUred

ABSTRACT
THE EFFECTS OF CHRONIC RESTRAINT STRESS

ON REPRODUCTIVE CONTROL MECHANISMS
IN THE LABORATORY RAT

BY

Sandra M. Wood

Effects of chronic restraint stress on serum LH,
prolactin and testosterone in male rats, serum LH in
female rats, and pituitary responsiveness to LHRH in both
male and female rats were determined in young three to five
month old Long—Evans rats. Serum samples were taken under
light ether anaesthesia via suborbital sinus puncture.
Samples were taken before and after two hours of restraint
stress on the first day, the seventh day and the thirteenth
day of stress. in vivg pituitary responsiveness was
measured prior to and after 10 or 12 days of restraint
stress. Following a zero time blood sample rats were
injected (i.v.) with 100 or 500 ng LHRH and blood samples
taken 15, 30 and 60 minutes or 15 and 45 minutes post-
injection. Following two weeks of restraint stress the
rats were decapitated and their pituitaries challenged
with either 0, 5, 25, 50 or 100 ng LHRH. After four hours

of incubation the media was decanted and the individual

pituitary he
used to meas
pituitary L}
pituitary hc
from control
enized and a
Seri
dose than t1
restraint S.
alter pitui
AlthOUQh tw
re5ponse to
rats, the p
did not Cha
second expe
response ma
groups rece
LH values f
control grc
Pituitary's
in male rat
as Seen as
IESpOnsE.
Pit
or LHRH Sti

rats.

LHr

Sandra M. Wood

pituitary halves homogenized. The incubation media was
used to measure pituitary LH and prolactin release, and
pituitary LH and prolactin content was measured from the
pituitary homogenate. In addition individual hypothalami
from control and stressed male and female rats were homog-
enized and assayed for dopamine and norepinephrine.

Serum LH response was greater to the 500 ng LHRH
dose than to the 100 ng LHRH dose in male rats. The
restraint stress treatments in the male groups did not
alter pituitary release of LH when stimulated with LHRH.
Although two weeks of stress decreased LH release in
response to 500 ng LHRH injection in one group of female
rats, the peak serum LH response to 500 ng LHRH injection
did not change between control and stressed females in the
second experiment. It was noted that the duration of the
response may be different in control and stressed female
groups receiving LHRH. The 45 minute postinjection serum
LH values from the stress animals were only one half of the
control group values. These results suggest that the
pituitary's ability to release LH is unaffected by stress
in male rats, while the response is altered in female rats
as seen as a decrease in peak release or duration of
response.

Pituitary content of LH was not affected by stress
or LHRH stimulation in either the stress or control male

rats. LH release from incubated pituitaries was stimulated

by LHRH, bu
the restrai
[i179 LHRH d
unaffected
pituitary h
cantly more
release of
restraint 5
data Suppor
that the pi
chronic Stri
Alt}
Period Was ..
Prestress a:
of the trea-
than COIltro
two WerS 0

fOlleing t‘

Sandra M. Wood

by LHRH, but the amount of LH released was unaffected by
the restraint stress. These data support that of the in

vivo LHRH data for male rats. Pituitary content was

 

unaffected by LHRH stimulation in female rats, but the
pituitary halves from the stress rats contained signifi-
cantly more LH than the control rats. LHRH stimulated
release of LH from the pituitary halves and chronic
restraint stress had no effect on pituitary release. These
data support that of the in yiyg LHRH data for female rats
that the pituitary LH control mechanisms may be altered by
chronic stress.

Although serum LH following the first restraint
period was found to be variable, LH was reduced in both the
prestress and poststress serum samples taken on day seven
of the treatment. Prestress serum LH levels remained lower
than control group LH in two of the three male groups after
two weeks of restraint treatment and were further reduced
following two hours of stress.

Changes in serum LH in response to the first
stress period in female rats depended upon the stage of
the estrous cycle. Resting serum LH was dramatically
reduced in female rats following one week of stress and was
unchanged after two hours of stress. Serum LH continued
to be low following two weeks of restraint stress.
Restraint stress also blocked ovarian cyclicity. Stressed
rats had persistent leucocytic vaginal cytology throughout

the treatment.

Pi'
prolactin .
in male ra

Se
LHRH injec
stress res
and was un
stress was
sterone af
data sugge
alters tes

Do
thalamus w
COHtr01 ma

thalamic C

Bo

Sandra M. Wood

Pituitary prolactin content and release and serum
prolactin concentrations were not altered by chronic stress
in male rats.

Serum testosterone increased in response to 500 ng
LHRH injection in control animals. Following two weeks of
stress resting serum testosterone was significantly reduced
and was unchanged by LHRH stimulation. Chronic restraint
stress was found to significantly decrease serum testo-
sterone after one and two weeks of restraint stress. These
data suggest that the stress-induced decrease in serum LH
alters testosterone release at the testes.

Dopamine and norepinephrine content of the hypo-
thalamus were not significantly different in stress and
control male rats. Stress also had no effect on hypo-
thalamic content of norepinephrine in female rats.

Both male and female stress groups lost significant
amounts of weight during the stress treatment.

These results indicate that chronic restraint
stress can interfere with reproduction in male and female
rats. Chronic restraint stress decreased serum LH in male
and female rats which was manifest as suppressed serum
testosterone in male rats and persistent diestrous vaginal
cytology in female rats. Chronic restraint stress had no
effect on LHRH stimulation of LH release suggesting that
the primary effect of stress on reproduction occurs in

the nervous system, presumably at the hypothalamus.

THE EFFECTS OF CHRONIC RESTRAINT STRESS
ON REPRODUCTIVE CONTROL MECHANISMS

IN THE LABORATORY RAT

BY

' C

Sandra M: Wood

A THESIS

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

MASTER OF SCIENCE
Department of Physiology

1976

TABLE

LIST OF TABLES . . . . .
LIST OF FIGURES . . . . .
INTRODUCTION . . . . . .

LITERATURE REVIEW . . .

General Considerations

Endocrine Control Mechanisms
Stress Effects on Reproduction
Stress Index Identification
Control of ACTH Secretion
Control of LH Secretion
Control of Prolactin Secretion
Role of Catecholamines in Controlling
Hypothalamo-Hypophyseal Secretion

MATERIALS AND METHODS .

Experimental Animals .
Blood Collection . . .
Restraint Stress . . .
Weights . . .

Radioimmunoassay for LH and

OF CONTENTS

Prolactin

Competitive Binding Assay for Testosterone

Catecholamine Assay .
Experimental Design .
Definition of Terms .

In Vivo LHRH Challenge
In Vitro LHRH Challenge

Hypothalamic Extract Preparation .

Anterior Pituitary Collection and

Incubation .

Plasma LH, Prolactin and Testosterone
Response to Stress

Statistical Analysis .

ii

Page

iv

11
15
16
22
28

29
33

33
33
34
34
34
37
39
40
42

43
43

44

44

45

46

Page

RESULTS . . . . . . . . . . . . . . . . . . . . . . . 47
LB Data . . . . . . . . . . . . . . . . . . . . . 47

In Vivo Pituitary LHRH Challenge . . . . . . . 47

In Vitro Pituitary LHRH Challenge . . . . . . 57

Stress Effects on Serum LH . . . . . . . . . . 62

PRL Data 0 O O O O O O C O O O O O O O O O O O O O 72

In Vitro Pituitary LHRH Challenge . . . . . . 72
Stress Effects on Serum Prolactin . . . . . . 75

Testosterone Data . . . . . . . . . . . . . . . . 75
Serum Testosterone Response to LHRH
Challenge . . . . . . . . . . . . . . . . . 75
Effect of Stress on Serum Testosterone . . . . 80
Catecholamine Data . . . . . . . . . . . . . . . . 8O
Vaginal Cytology Data . . . . . . . . . . . . . . 80
Chronic Stress Effects on Body Weights . . . . . . 84
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 86

LIST OF REFERENCES . . . . . . . . . . . . . . . . . . 98

iii

Table

1. The Eff
on Hy
Young

2. The Eff
orl Bc:
Rats

LIST OF TABLES
Table Page
1. The Effects of Chronic Restraint Stress
on Hypothalamic Catecholamines in
Young Female and Male Rats . . . . . . . . . . 83
2. The Effects of Chronic Restraint Stress

on Body Weight of Male and Female
Rats I O O O O O O O O O I O O O O O 0 O O O O 85

iv

Figure

1.

10.

11.

12.

13.

LIST OF FIGURES

Effects of 100 ng and 500 ng LHRH on
serum LH in young male rats--
CoRoo" -I o o o o o o o o o o o o o o 0

Effects of 500 ng LHRH on serum LH in
young male rats-~C.R.C? -II and
CoRoOw-III o o o o o o o o o o o o o o

The effects of LHRH on serum LH con-
centrations in young female rats . . . .

The effect of LHRH on pituitary content
on LH in young male rats . . . . . . . .

The effect of LHRH on pituitary release
of LH in young male rats . . . . . . . .

The effect of LHRH on pituitary content
of LH in young female rats . . . . . . .

The effect of LHRH on pituitary release
of LH in young female rats . . . . . . .

The effect on chronic restraint stress
on serum LH in young male rats . . . . .

The effects of stress on serum LH in
young female rats . . . . . . . . . . .

The effects of stress on pituitary content
of prolactin and ability to release
prolactin in young male rats . . . . . .

The effect of stress on serum prolactin
in young male rats . . . . . . . . . . .

The effects of 500 ng LHRH on serum
testosterone in young male rats . . . .

The effect of stress on serum testosterone
in young male rats . . . . . . . . . . .

Page

49

52

55

59

61

64

66

68

7O

74

77

79

82

Acx
hypothalam<
hypothalam<
actions. 1
considered
the adenoh:
understand:
ComPonents
is often c:
Stl‘esses s-
intehsity.
StudiEd in
Chronic St

Th
the EffeC t
(phygiCal
of the lab
investigat
(four to f

normal heu

INTRODUCTION

Acute stress has been largely used to study the
hypothalamo-hypophyseal—adrenal axis as well as the
hypothalamo-hypophyseal-gonadal axis and their inter-
actions. Although many investigators have individually
considered the effect of stress on serum concentrations of
the adenohypOphyseal, adrenal and gonadal hormones, our
understanding of the effect of stress on central system
components remains minimal. The literature in this field
is often confusing and conflicting due to the variable
stresses studied and inadequate measurement of stress
intensity. While acute stress effects have been extensively
studied in laboratory rodents, domestic animals and humans,
chronic stress has been less well studied.

The objective of these experiments was to determine
the effect of a defined stressor of high intensity
(physical restraint) on gonadal control system components
of the laboratory rat. The rat was chosen for this
investigation because of its short reproductive cycle
(four to five day estrous cycles in the female). Also

:normal neuroendocrine control of gonadotrOpin and prolactin,

as well

in this

as well as ovulation, has already been fully characterized

in this species.

LITERATURE REVIEW

General Considerations

 

Two different organ systems exist in metazoa for
the integration of bodily functions, the nervous system and
the endocrine system (Frings and Frings, 1970). These two
control systems have developed during the course of
evolution, and exhibit some characteristics complementary
to each. The nervous system occurs in most multicellular
organisms, even coelenterates, and plays an essential role
in the control of adaptation to changes in both internal
and external environments (Dethier and Stellar, 1970).
Neurons have both an excitatory and secretory nature
(Mountcastle, 1974). Excitatory neurons can generate
action potentials as a result of their excitation and
rapidly transmit the signal through the nerve fiber to
other cells. Secretory neurons produce various kinds of
specific information-carriers called neurohormones
(Dethier and Stellar, 1970; Frings and Frings, 1970).
These chemical messengers are liberated from fiber endings
into the body fluid and exert specific effects on other
cells. The neurosecretory system shows characteristics

between the nervous and endocrine system. In invertebrates

neurosec
to carry
more con
certain
Dethier
and rele
possesse
the com
tissues
Control
While n
(Mountc
time (16
and Can

duced ;

neurosecretory neurons and excitatory neurons are sufficient
to carry out regulatory function, but as organisms become
more complex, the endocrine system develops to regulate
certain bodily functions (Inoué and Sekiguchi, 1973;
Dethier and Stellar, 1970). These endocrine organs produce
and release hormones into the body fluid. Each hormone
possesses a specific chemical composition which eliminates
the contamination of information as well as specific
tissues sensitivity to the specific chemical agents help
control selection (Usher, Kasper, and Birmingham, 1967).
While neural transmission and control is very rapid
(Mountcastle, 1974), endocrine control is followed by a
time delay and a longer lasting effect (Nequin, Alerez,

and Campbell, 1975). Hormonal information can be trans-
duced into neural information, and neural action potentials
into chemical signals. Hormones may also modify the
excitability of neurons as well as neural input may alter
hormone secretion (Inoué and Sekiguchi, 1973; Allen, Allen,
Greer, and Jacobs, 1973).

Neuroendocrinology deals with the interaction of
the nervous and endocrine systems in the control of body
functions and in the adaptation of an individual in its
environment (Donovan, 1970). Intensive study of the
relationship between nervous and endocrine systems in
mammals has been largely directed toward analysis of the

factors involved in the neural control of the pituitary

gland. Meaningful study of brain endocrine relationship
began with this century (Meites, Donovan, and McCann, 1975).
Although the testicular transplantation experiments by
Berthold in 1849 gave information indicative of internal
secretion it was not until 1902 that Bayless and Starling
prOposed the name "hormone" to describe the role of
secretion in the control of pancreatic secretion (Donovan,
1970; Mountcastle, 1974). About this time the neuro-
hypophysis was associated with the fast acting and short
lived actions of pituitary extract effects on the cardio-
vascular system, the uterus and mammary glands. It was not
until Philip Smith developed a technique for removal of the
hypophysis without damaging the hypothalamus that physio-
logical function was described for the adenohypophysis
(Brooks, 1975). In 1927 Smith showed that hypophysectomy
resulted in retarded growth and atrophy of the gonads,
thyroid gland, adrenal cortex, liver, spleen and kidneys.
These changes could be reversed by crude pituitary extract
injections or by implanting pituitary tissue (Donovan,
1970; Mangili, Motta, and Martini, 1966). In 1936 Marshall
concluded that many external influences including emotional
stimuli could modify the sexual cycle by acting on the
pituitary gland through the central nervous system
(Donovan, 1970; Neville and Neathery, 1974; Chatterton,
Chien, Ward, and Miller, 1975). Believing the anterior

pituitary to be under direct neural control, scientists

began t
and ide
examina
over th
In 1947
between
hypophy
1970 ; M¢
that fo,
anterio;
regener;
barrier
hYpofum
blood re
proPert;
body 0t]
Supply 1
this tYl
hYpotha;

began to search for the connecting link. Effort to locate
and identify nerve tracks failed (Jacobsohn, 1975) and
examination by the electron micrOSCOpe ended the dispute
over the secretomotor innervation of the anterior pituitary.
In 1947 Green and Harris found evidence that the pathway
between the hypothalamus and anterior pituitary was the
hypophyseal portal vessels (Green and Harris, 1947; Donovan,
1970; Mountcastle, 1974; Jacobsohn, 1975). They found

that following transection of the pituitary stalk that
anterior pituitary function returned as the portal vessels
regenerated. When regeneration was prevented by placing a
barrier at the cut stalk, gonadal atrophy and pituitary
hypofunction continued. It was then hypothesized that the
blood reaching the pituitary contained some special
properties. Pituitary tissue transplanted to areas of the
body other than the median eminence reestablished its blood
supply but not its function (Hinsey, 1975). Evidence of
this type supported the neurohumoral theory that the
hypothalamus releases chemical agents into the portal
vessels and exert their effect on the cells of the anterior
pituitary (Donovan, 1970). Confirmation of this hypothesis
came from experiments using hypothalamic extracts to alter
adenohypophysis function. Appropriate hypothalamic extracts
were found to release either follicle-stimulating hormone
(FSH—RH) (Johansson et al., 1973), luteinizing hormone

(LHRH) (Matsuo et al., 1971), adrenocortocotrophic hormone

(CRH) (Shally et al., 1960; Reniker, Sieber, and Rittel,
1972), or thyrotrophic hormone (TRH) (Bowers et al.,
1970; Burgus, 1970), or to inhibit prolactin (PIF) release

(Meites, 1972).

Endocrine Control Mechanisms

 

Endocrine control mechanisms have been examined
theoretically and models designed to graphically explain
neuroendocrine control systems (Donovan, 1970; Dethier
and Stellar, 1970). The model consists of the higher
centers of the CNS, the hypothalamus and its releasing or
inhibiting factors, the anterior pituitary and its hor-
mones, target tissues and their hormones. The effect of
the target tissue hormones at the CNS, hypothalamus and
anterior pituitary completes the control system. Both
negative and positive feedback have been demonstrated at
the anterior pituitary (Price, 1975). When rats were
hypophysectomized the gonads atrophied, and administration
of fresh pituitary extracts restored the gonads to normal.
Implantation of mature rat pituitaries into the median
eminence of immature females causes precocious maturity
(Price, 1975). These findings established a link on the
chain of evidence that provided the background for neuro-
endocrine control. In the 19205 it was established that
the gonads secrete estrogens, progesterone and androgens.
It was also demonstrated that the anterior pituitary gland

secreted gonadotrOpins that stimulated the gonads to

secrete sex hormones (Price, 1975; Bogdanove, 1972). Daily
injections of estrogen or testosterone were found to have
negative effects upon bull testes. Price interpreted this
data that estrogen and androgens must act to inhibit
pituitary gonadotropins. Negative feedback of adrenal
steroids was demonstrated when cortisol implants blocked
adrenocortical function such as the stress induced release
of adrenal steroids (Sawyer, 1975). While negative feedback
is the most common endocrine influence on pituitary hormone
release positive feedback can occur with ovarian hormones
(Price, 1975; Hohlweg, 1975; Harris, 1964). Estrogen can
trigger secretion of LH from the anterior pituitary during
follicular development which causes ovulation and stimu-
lates formation of corpora lutea. Single injections of
estradiol to immature female rats resulted in corpora lutea
formation (Hohlweg, 1975). This positive feedback effect
is specific for females, as injection of estrogen into male
.rats does not cause secretion of LH. Hormonal negative
feedback has also been demonstrated at the level of the
hypothalamus. Hohlweg (1975) found that pituitaries
transplanted to kidney capsules showed no signs of alter-
ation after castration. If pituitaries from castrated
animals were transplanted to the kidney capsule it was
impossible to observe castration cells in the anterior
pituitary after three weeks. These data indicated that the

nervous center must monitor the gonadal steroid serum

concentrations and can alter gonadotropin secretion.
Experiments of Harris and Jacobsohn (1952) showed a
decrease in LH activity and gonadal steroids when rat
pituitaries were removed and placed under the kidney cap-
sule. This condition was alleviated when the anterior
pituitary was reinstated just below the median eminence.
This confirmed the probable existence of a neurohormone
which travels to the anterior pituitary by way of the
hypophyseal portal vessels. Hypothalamic lesions were
found to cause dystrophic changes in both the adrenal
cortex and the gonads, especially lesions placed in the
medioventral and rostral regions (Ingram, 1975). The first
stereotaxically-placed implants of crystalline estradiol
and testosterone were placed in the rat brain by Lisk
(1960). He was able to differentiate a basal hypothalamic
area where steroids induced gonadal atrophy and an area
rostral to the preoptic area that influenced sexual
behavior. Sawyer (1967) found, in both rabbits and dogs,
that gonadal steroids induced gonadal atrophy if placed at
the basal hypothalamus but had no effect if placed at the
pituitary or other areas of the brain. The effective site
of estrogen implantation at the hypothalamus is the same
site that electrical stimulation induced ovulation and
lesions caused ovarian atrophy (Sawyer, 1963). While
intrapituitary estrogen had no effect on the ovary or the

uterus, indicating no steroid feedback at the pituitary,

10

it did activate the mammary glands, indicating prolactin
release (Sawyer, Hilliard, Kenematsu, Scaramuzzi, and
Blake, 1974). Bioassays of pituitary prolactin and LH
showed that intrahypothalamic estrogen interferred with
synthesis and release of pituitary LH while intrapituitary
estrogen resulted in release and depletion of pituitary
prolactin (Kawakami and Sawyer, 1967). Deaffrentation of
rostral and preoptic areas of the hypothalamus using the
technique of Halaéz were found to eliminate cyclic ACTH and
gonadotropin secretion, thus eliminating diurnal changes in
levels of adrenal steroids and cyclic ovulation (Sawyer

and Gorski, 1971).

Even with the absence of target organ hormone
pituitary trophic hormones can still influence hypothalamic
activity, indicating a short-100p feedback (Beyer and
Sawyer, 1969). Hypothalamic implants of LH decreased
multiunit activity when placed in the preoptic area and
ventromedial area. ACTH implants were found to increase
multiunit activity when placed in the median eminence in
adrenalectomized rats, while dexamethasone implants
depressed the activity (Sawyer, 1970).

Besides the feedback from the adrenal steroids on
ACTH secretion at the pituitary, and gonadal steroid
effects on LH and FSH, these systems can interact with
each other. In 1939 Selye found that stress induced

adrenal corticoid activity was accompanied by gonadal

atrophy. In
secretion an
on social be
decrease in
(Scott, 195'
animals bec
accompanied
(Scott, 195
rate of mo:

SteHar. is

11

atrophy. In 1963 Schreiber found stress increased ACTH
secretion and decreased gonadotropin release. Experiments
on social behavior with mice and hamsters showed a rapid
decrease in animal numbers following a peak population
(Scott, 1958; Dethier and Stellar, 1970). Submissive
animals became thin, lost hair and often had skin lesions,
accompanied by adrenal hyperplasia and gonadal atrophy
(Scott, 1958). These endocrine disorders lead to a high
rate of mortality, and reduced fertility (Dethier and

Stellar, 1970).

Stress Effects on Reproduction

 

A variety of experiments have shown a relationship
between stress or increased adrenocortical function and the
reproductive control system. Surgical stress was found to
decrease serum LH and FSH in humans (Charters, Odell, and
Thompson, 1969) and ACTH could block PMS induced ovulation
in immature rats (Hagino, Watanabe, and Goldzeiher, 1969).
Injection of hydrocortisone, prednisolone or ACTH during
the follicular phase of the estrous cycle in the sow
delayed estrous one to three days and shortened the estrus
period from three days to one day (Liptrap, 1970).
Administration of ACTH in man was found to decrease serum
testosterone by inhibiting LH release from the pituitary
(Rivarola, Saez, Meyer, Jenkins, and Migeon, 1966). Ajika
et a1. (1972) found pentobarbitol blocked ether stress-

induced LH elevation in rats, but administration of

12

hypothalamic extracts or LHRH stimulated LH release in
these rats. Restraint stress before ovulation and mating
in rats had no effect on litter size but did delay ovula—
tion by three days (Euker and Riegle, 1973).

Reproductivity in ewes was affected by excessively
high or low ambient temperatures during the breeding
season, as exhibited by delayed estrous cycles and
increased embryonic mortality (Neville and Neathery, 1974).
Restraint stress during the first five days of pregnancy
or the fifth through twelfth day of pregnancy of the rat
interrupted more than 50 percent of the pregnancies.
Animals that remained pregnant had normal litter numbers.
This all or none response to stress was further substanti-
ated by the effects of restraint stress on the afternoon
of proestrus (McKay et al., 1976) where stress either
totally blocked ovulation or had no effect.

Although Euker et a1. (1975) found ether stress,
gentle handling and blood sampling increases serum LH in
intact male rats, the same treatment resulted in decreased
serum LH in castrated male rats. Although rats treated
with low doses of dexamethasone could still respond to the
handling stress with increased serum corticosterone and
LH, dexamethasone treatment reduced the magnitude of the
stress-induced response for both hormones.

In another experiment, it was shown that serum LH

of intact male rats remained unchanged following two hours

of restra:
diestrus <
restraint
estrus ra1
contrast 1
female ra1
less of t}
size diffs
between la
experiment

86

Stress (We

13

of restraint stress, as did serum LH in female rats during
diestrus or estrus (Euker et al., 1973). Two hours of
restraint stress was found to decrease serum LH in pro-
estrus rats (Euker et al., 1973; McKay et al., 1976). In
contrast to these findings Neill (1970) found serum LH in
female rats unaffected by ether or bleeding stress regard—
less of the stage of the estrous cycle. These data empha-
size differences in stress effects on hormone release
between laboratories and the reproductive status of the
experimental animals.

Serum prolactin concentration is also affected by
stress (Wakabayashi et al., 1971). Acute stress such as
handling, ether exposure and serial blood sampling increases
serum prolactin within 2 to 4 minutes in male rats (Euker,
Meites, and Riegle, 1975; Bellinger and Mendel, 1975).

Two hours of restrint stress increased serum prolactin in
male rats, in diestrus female rats (Euker, Meites, andx
Riegle, 1973; Riegle and Meites, 1976), and in ovariecto-
mized rats (Ajika et al., 1972; Riegle and Meites, 1976).
Both dexamethasone (Harms, Langlier, and McCann, 1975;
Euker, Meites, and Riegle, 1975) and pentobarbitol (Ajika
et al., 1972) can block the stress-induced prolactin
increase in male and ovariectomized rats. Serial ether
stress decreased serum prolactin in proestrous rats, and
had no effect on estrous or diestrous rats prolactin con-

centrations (Euker, Meites, and Riegle, 1975; Riegle and

Meites, 1‘.
serum prol
during prc
or on the
hours of 1
proestrous
Meites, 15

St
events the
has been 6
damage or
1973). Rt
serial ble
are but a
RiEgle' IS
and Brown!
stress Eff
haVe been

restraint

rats, Whi]

HEites ’ ls

ShOwn to C

(Euker

0 ME
importamt

l4

Meites, 1976). Serial blood sampling was found to decrease
serum prolactin on the afternoon of estrus as well as
during proestrus, and had no effect on rats during diestrus
or on the morning of estrus (Riegle and Meites, 1976). Two
hours of restraint stress decreased serum prolactin in
proestrous and estrous rats (Euker et al., 1973; Riegle and
Meites, 1976).

Stress is a term used to describe a variety of
events that can illicit a multitude of responses. Stress
has been defined as a collection of diverse stimuli which
damage or potentially damage the organism (Allen et al.,
1973). Ether stress, handling stress, novel environment,
serial bleeding, surgery, leg break and restraint stress
are but a few of the different stressors used (Euker and
Riegle, 1973; Baldwin, Colombo, and Sawyer, 1974; Seggie
and Brown, 1975; Blake, 1975) in experiments to study
stress effects on reproduction. These different stresses
have been found to cause conflicting data, as two hours of
restraint stress increases serum prolactin in diestrous
rats, while serial ether stress had no effect (Riegle and
Meites, 1976). In addition restraint stress has been
shown to decrease serum LH in proestrous rats, have no
effect on serum LH male, diestrous or estrous female rats
(Euker, Meites, and Riegle, 1973). Therefore it is

important for investigators to carefully define stresses

so thl

ments

ACTH I
adrene
1973;

eral r
mit ar
Stimu]
trollj
et a1.
main c

1973).

15

so their different effects can be compared between experi-

ments and between various investigators.

Stress Index Identification

 

The most accepted index of stress is the release of
ACTH from the adenohypophysis followed by an increase in
adrenal glucocorticoids secretion (Brodish and Redgate,
1973; Donovan, 1970). The central nervous system, periph-
eral nervous system and the autonomic nervous system trans-
mit and regulate stress-induced impulses to the hypothalamus
stimulating the hypothalamic-hypophyseal mechanisms con-
trolling ACTH release (Dethier and Stellar, 1970; Allen
et al., 1973). Stressors have been broken down into two
main categories, systemic and neural stress (Allen et al.,

1973).

I. Systemic stress
A. Internal environmental changes
1. physiological processes

hypotension
hypoglycemia
hypoxia
starvation
exercise

2. pharmacological substances

epinephrine ether
histamine parathione
ADH capsaicin
formalin

endotoxin

16

B. External environmental changes
external temperature

heat
cold

forced immobilization or restraint
II. Neural stress
A. Noxious stimulus
leg break
tourniquet
electric shock
vibration
burning
B. Light
C. Sound

D. Psychological stress--emotiona1 stress

Neurogenic stresses such as leg break or surgery
use neural pathways to the hypothalamus by way of the
peripheral nervous system to the spinal cord and central
nervous system while systemic stress act through the cir—'
culation and activates neural signals to the hypothalamus
and pituitary (Allen et al., 1973). Maximal stress is
found when both neurogenic and systemic pathways are

activated as seen with starvation and exercise.

Control of ACTH Secretion

 

Normally the adrenal glands secrete three groups
of steroids, glucocorticoids, mineralocorticoids, and
adrenal sex steroids (Mountcastle, 1974). Both stimulated

growth of the adrenal cortex at the zona fasciculata and

zona l
cortic
glucoc
(hydrc
man, c

glucoc

synthe
a smal
Studie
5 mini
adreng
Sparks
be I8c
measu]
ACTH a
of Ste

minute

l7

zona reticularis, causing synthesis and release of gluco-
corticoid steroids and adrenal androgens. Secretion of
glucocorticoids vary from species to species, as cortisol
(hydrocortisone) is the major glucocorticoid secreted in
man, dog, and guinea pig and corticosterone the main
glucocorticoid in the cat, rat, and mouse (Donovan, 1970).

The chemical structure of ACTH, characterized and
synthesized by Schwyzer and Sieber (1963), was found to be
a small polypeptide of 39 amino acid residues. Half life
studies have shown that 50 percent of ACTH is lost within
5 minutes postinjection in intact rats, 30 seconds in
adrenalectomized rats and 10 minutes in man (Evans,
Sparks, and Dixon, 1966). This indicates that there must
be regular pituitary release of ACTH in order to maintain
measurable levels of ACTH in the blood. The action of
ACTH at the adrenal cortex is very rapid as the release
of steroids into adrenal venous blood can be detected two
minutes following intravenous injection of ACTH (Evans et
al., 1966; Donovan, 1970).

Hans Selye (1936) initiated the concept of "stress"
as he found ACTH release from the anterior pituitary was
induced by a variety of stimuli and developed the theory
that naturally occurring stresses can cause "diseases of
adaptation." It is now clear that ACTH secretion fluctu-
ates in response to a vast number of stimuli which range

from psychological conditions such as anxiety, fear and

anger to
(Dethier
and Wodzi
Increases
found in
before ex
patients
gency war
0
exists be
and the s
adrenal c
and resul
1970; Tip
Pensatory
maintain
ACTH incr
ectOmY an
(Tiptaft'
Stimulus
above the
E
Parts Of
contrOl 8
formation

18

anger to cold, heat, and surgical stress or overcrowding
(Dethier and Stellar, 1970; Allen et al., 1973; Hearnshaw
and Wodzicka-Tomaszewska, 1973; Euker et al., 1973).
Increases in serum ACTH and adrenal steroids have been
found in peOple in stressful situations such as students
before exams, aircraft pilots, emotionally disturbed
patients and relatives accompanying patients at an emer—
gency ward of a hospital (Mason, 1968).

Observations attest to the fact that a balance
exists between the blood concentration of adrenal hormones
and the secretion of ACTH as chronic administration of
adrenal corticoids inhibits pituitary secretion of ACTH
and results in atrophy of the adrenal cortex (Donovan,
1970; Tiptaft, 1975). Removal of one adrenal causes com-
pensatory hypertrophy of the remaining adrenal in order to
maintain blood corticoid concentration (Mountcastle, 1974).
ACTH increases in the blood following bilateral adrenal-
ectomy and injection of corticosteroids can lower ACTH
(Tiptaft, 1975). These workers also found that noxious
stimulus applied to adrenalectomized rats increased ACTH
above the already elevated levels.

Electrical stimulation and lesioning of various
parts of the brain has demonstrated that higher centers
control secretion of ACTH. Stimulation of the reticular
formation in the midbrain can increase release of ACTH

(Taylor, 1969). Brown et a1. (1974) also found increase

in ACTH af
cingulate
monkeys.
hypothalam
and adrena
Martini, 1
Le
blocks Str
tested (Br
Birmingham
anterior c
COSterone
Schally' a
”sterone
lesions.
adrenergic
Was fOund
releaSe an:
blockel.) i]

19

in ACTH after stimulation of the amygdaloid nuclei, and
cingulate gyrus of the frontal lobe in dogs, rabbits and
monkeys. Stimulation of the septum, hippocampus, anterior
hypothalamus and the midbrain can show a reduction in ACTH
and adrenal hormones in most mammals (Mangili, Motta and
Martini, 1966).

Lesions in the tuberal and median eminence regions
blocks stress-induced ACTH release in all mammalian species
tested (Brodish, 1963). In 1967, Usher, Kasper, and
Birmingham found lesions in the lateral septal area or
anterior cingulate gyrus blocked stress-induced corti-
costerone release in rats, but Brown, Uhler, Seggie,
Schally, and Kastin (1974) found an increase in corti-
costerone release following stress in rats with septal
lesions. Control of ACTH release may be related to the
adrenergic system. Stimulation of the adrenergic system
was found to block surgically—induced corticosterone
release and injection of phenoxbenzamine (a-adrenergic
blocker) into the lateral ventricle was found to augment
the surgical stress response (Eisenberg, 1975). On the
other hand, intraventricular injection of norepinephrine
or dopamine had no effect on stress-induced corticosterone
release. Administration of 6-hydroxdopamine intracere-
brally (depletes brain catecholamines) was found to
decrease only norepinephrine in adult rats and norepine-

phrine and dopamine in immature rats. These depletions

20

had no effect on the basal diurnal periodicity of ACTH or
on responsiveness to stress (Kaplanski, van Delft, Nyakas,
Stoof, and Smelik, 1974). Other investigators have found
that catecholamines play an important role in ACTH secre-
tion (Ganong, 1972). Abe and Hiroshige (1974) hypothesized
that catecholamines may act to stimulate ACTH release but
depletion of catecholamines, especially norepinephrine, had
no effect on circadian rhythms or stress-induced release
of CRF, ACTH or corticosterone (Abe and Hiroshige, 1974;
Kaplanski et al., 1974). While it appears that brain
catecholamines plays a role in CRF-ACTH secretion, they are
not necessary for CRF, ACTH and corticosterone release in
response to stress. Perhaps catecholamines are only one
of the mechanisms by which CRF and ACTH are regulated and
when depleted another mechanism such as the cholinergic
system, compensates for it.

Although it is clear that the brain plays a role
in ACTH secretion, adrenal corticosteroids can also control
ACTH secretion through negative feedback. In rats adminis-
tration of adrenal corticoids can block the response to
stress (Sawyer, 1975). There is a linear relationship
between the amount of corticosteroid given and the
inhibition observed (Donovan, 1970). Synthetic gluco-
corticoids such as dexamethasone also block the stress

response (Euker, Meites, and Riegle, 1975).

Ac
areas witl
inhibit A<
to the tul
secretion
while sim:
at blOCkil
1966). C.
rats betw.
effective
aCtion de'
t0 the pi
loop feedj
MESS and J
implanted
endogenOul

lng adreI-l1

21

Adrenal hormone implants have been used to locate
areas within the brain where corticosteroids feedback to
inhibit ACTH release. Application of cortisone in agar
to the tuberal region of the hypothalamus inhibits the
secretion of adrenal corticosteroids in rats and cats,
while similar implants in the pituitary proved ineffective
at blocking ACTH release (Mangili, Motta, and Martini,
1966). Corticosterone implanted in the hypothalamus of
rats between the optic chiasm and median eminence was most
effective at suppressing ACTH release. The inhibitory
action decreased as the implant placement was made closer
to the pituitary stalk (Smelik, 1969). Evidence for short-
loop feedback for ACTH at the hypothalamus was found by
Mess and Martini (1968). These workers showed that ACTH
implanted into intact and adrenalectomized rats depressed
endogenous release of ACTH, and that CRF secretion follow-
ing adrenalectomy was blocked by ACTH injection.

Corticotropic-releasing factor (CRF) was found in
the median eminence (Shally et al., 1960; Riniker, Sieber,
and Rittel, 1972). At one time vassopressin (ADH) was
believed to be CRF, as administration of ADH increased the
release of ACTH from the pituitary (McCann and Porter,
1969). However rats with diabetes insipidus secrete ACTH
and corticosterone following stress. Also it was found
that ethanol can block secretion of ADH without blocking

ACTH release (Purves and Sirett, 1967). While many agents

act
Hiro
neit
for

and

22

act to release ACTH, such as catecholamines (Abe and
Hiroshige, 1974), and ADH (McCann and Porter, 1969)

neither of these substances have been shown to be necessary
for stress-induced ACTH and plasma corticosterone (Fuxe

and Hokfelt, 1969; Abe and Hiroshige, 1974).

Control of LH Secretion

 

Although there remains some disagreement about the
roles of the gonadotropins, it is now generally accepted
that FSH stimulates growth of the follicle and LH is
responsible for maturation and ovulation of the follicles.
Ovulation occurs when LH levels are increased in remales
whose ovaries contain mature follicles. LH is also respon-
sible for production and secretion of estrogen from the
maturing follicle and it is also necessary for progesterone
secretion from corpora lutea in some species. In males FSH
acts at the first stages of spermatogenesis and hyper-
trophies the seminiferous tubules. LH stimulates androgen
production and indirectly supports spermatogenesis
(Mountcastle, 1974; Ganong, 1971).

The events taking place during the four day estrous
cycle of the rat have been summarized by Swartz (1968) as
follows:

1. estrus—new follicles begin growing, possibly
stimulated by the FSH and LH surge the day before

2. metoestrus and the first day of diestrus-follicle
growth continues

3. second day of diestrus-LH begins to stimulate
release of estrogen

sc

pr

5 cu

of
Kobayashi ,

estrogen i
followed t
PIOgesterc
1972). 1&3

Ir
just follc
the LH pee
(Tamada, '1
(1972) be]
triggers t
decreaSed

conjugalted
al., 1973;

This is 311

found an F

23

4. proestrus-estrogen peak followed by LH and FSH
surge between 2:00 and 7:00 p.m. Follicles begin
preovulatory swelling
5. ovulation at approximately 2:00 a.m. the morning
of estrous.
Kobayashi, Hara, and Miyak (1968) found ovarian venous
estrogen increased the morning of proestrus which was
followed by increased LH later in the afternoon. Serum
progesterone increased in parallel with LH (Swerdloff,
1972). 1972

In women LH is elevated for 24 hours during or
just following estrogen elevation. In all cases studied
the LH peak never occurred before the estrogen peak
(Tamada, Tsukui, Araki, and Matsumoto, 1973). Swerdloff
(1972) believes estrogen synergizing with progesterone
triggers the preovulatory peak. Plasma levels of LH
decreased within 2 to 4 hours following injection with
PremariHG>(an equine derived hormone complex including
conjugated estrogens) in ovariectomized women (Tamada et
al., 1973; Swerdloff, 1972). Within 24 hours postinjection
LH increased, reflecting the decrease in injected estrogen.
This is supported by the data of Odell et a1. (1968) who
found an FSH-LH peak within 24 hours after a single
injection of progesterone. This demonstrates negative
feedback mechanism between LH and estrogen. Injection of
pentobarbital prior to ovulation can block ovulation in

rats (Swartz, Cobb, Talley, and Ely, 1976; Tamada et al.,

1973). In experiments using antisera to LH and estrogen,

24

pentobarbital was found to block progesterone secretion
but not estrogen secretion. Even in the presence of anti-
LH, LH can be detected in the blood, but not at concentra-
tions that will cause ovulation. Estrogens ability to
trigger LH may be a threshold phenomenon or the dynamic
rate of change of estrogen may trigger LH release (Tamada
et al., 1973).

Gonadotropin secretion is controlled by higher
centers. The pituitary secretes little FSH or LH following
pituitary stalk lesioning (Donovan, 1970). Ovulation could
be induced by stimulation of the medial preoptic area and
anterior hypothalamic area, although lesions of the medial
preoptic area will block ovarian cycles, severing the
posterior connections of the hypothalamus does not alter
the estrous cycle in the rat (Everett, 1964). Isolation of
the hypothalamus by use of the Halasz knife, had no effect
on male rat testes histology, but female rats estrous
cycles ceased (Butler and Donovan, 1969). If the supra
chiasmatic nuclei are included in the Halasz knife isolated
hypothalamic island, persistent vaginal cornification
(estrus) was seen, otherwise persistent corpora lutea
(anestrus or diestrus) is observed.

A two level concept of the control of gonadotropins
secretion has been theorized. A basal level of secretion
is mediated by the median eminence while the preoptic area

is necessary for the production of gonadotropin surges in

female

ing tec
sized c
suprach

(Mess a

gonadot
of stud
steroid
arcuate
gonads
implan1
male r;
neceSS;
ovarie.
tion a
serum

and Ski
hYpoth

and Ps

inject

25

female rats (Butler and Donovan, 1969). Using the lesion-
ing technique, FSH-RH has been demonstrated to be synthe-
sized close to the paraventricular nuclei, and LHRH at the
suprachiasmatic-preoptic and arcuate ventomedial region
(Mess and Martini, 1968).

Estrogen, progesterone and testosterone can inhibit
gonadotropin secretion (Yagi and Sawaki, 1973). A number
of studies have provided evidence for feedback effects of
steroids at the hypothalamus. Implants of estrogen in the
arcuate nuclei of the hypothalamus caused atrophy of the
gonads in rats (Lisk, 1960). Testicular atrophy follows
implantation of testosterone at the median eminence in
male rats (Davison, 1967). The anterior hypothalamus is
necessary for compensatory hypertrophy following unilateral
ovariectomy (Koves and Halész, 1969). Estrogen implanta-
tion at the hypothalamus was found to suppress the elevated
serum LH following ovariectomy (McCann et al., 1964; Gay
and Sheth, 1972). Ovariectomized rats with anterior
hypothalamic lesions did not have elevated serum LH
(Taleisnuk, 1961).

Estrogen can also stimulate the release of LH
(Davison, 1969). A single injection of estrogen can
facilitate LH secretion in prepubertal, adult, pregnant,
and pseudopregnant rats (Yagi and Sawaki, 1973). Daily

injection of estrogen can cause an LH and FSH surge on

the fifth
(Swerdlof

F
hypothale
feedback
in contac
gonadotrc
Estrogen
atrOphy l
hYPOthala
thalamic
ovariecu
Chiasmat:
compenSa‘
Halész e.
also bEe;
hypothal.
medial p;
areas an
tropin rm
studj.es :
estrogen
hyPOthali
negatin

0C

26

the fifth day of injection in ovariectomized rats
(Swerdloff, Jacobs, and Odell, 1972).

Receptor cells for estrogen must be present in
hypothalamic-hypophyseal cells for positive and negative
feedback mechanisms to exist. These receptors need to be
in contact with neurosecretory neurons that secrete
gonadotropin releasing factors in the median eminence.
Estrogen implants in the arcuate nucleus cuases ovarian
atrophy (Lisk, 1960). Estrogen implants at the anterior
hypothalamus or medial basal hypothalamus decrease hypo-
thalamic LHRH activity and suppressed LH release following
ovariectomy (McCann et al., 1964). Lesions in the supra-
chiasmatic area and medial basal hypothalamus abolished the
compensatory hypertrophy following unilateral ovariectomy
Halasz et al., 1967). Estrogen responsive neurons have
also been found in the medial preoptic and medial basal
hypothalamus in castrated female rats (Yagi, 1973). The
medial preoptic, anterior and medial basal hypothalamic
areas are involved in estrogens negative feedback on gonado-
tropin release (McCann et al., 1964). Single unit response
studies showed decreases in neural firing rates following
estrogen implantation at the preoptic and medial basal
hypothalamic areas (Yagi, 1973).

There is also evidence that the gonadotropins have
negative feedback at neural control centers, independent

of gonadol steroids as intermediaries. FSH and LH implanted

into the
content
has the

preoptic

effects
the Sex
1975; Ne
Christi;
inhibit.
serum_1J
Restrai:
estrous
in proe
and saw
another
female

rats Pr‘

LH. LH

rats an
treatme
reStrai
EVidEnC
COrticO

1976) ‘

27

into the hypothalamus depresses the pituitary LH and FSH
content (Motta, Franchini, and Martini, 1969). LH also
has the ability to decrease neuronal unit activity in the
preoptic area and basal medial hypothalamus (Sawyer, 1970).
While stress is known to increase ACTH release its
effects on gonadotropin secretion may partially depend on
the sex of the animal involved (Euker, Meites, and Riegle,
1975; Neill, 1970; Shaar et al., 1975). In 1965,
Christian, Lloyd, and Davis suggested LH secretion was
inhibited by ACTH in female mice. Ether stress increased
serum LH in intact male rats (Euker et al., 1975).
Restraint stress had no effect on serum LH in male rats or
estrous or diestrous female rats, but decreased serum LH
in proestrus rats (Euker et al., 1973). Baldwin, Colombo,
and Sawyer (1974) found moving rats from one room to
another had no effect on serum LH of proestrus rats; or
female rats primed with PMS and HCG, while ovariectomized
rats primed with estrogen exhibited an increase in serum
LH. LH is released in a pulsatile manner in ovariectomized
rats and surgery, injection of ACTH, or corticosterone
treatment does not alter this pulsatile release of LH but
restraint stress will block it (Blake, 1975). Further
evidence that stress does not need to act through ACTH or
corticosteroids to decrease gonadotropins is observed in
adrenalectomized female rats (McKay, Wood, and Riegle,

1976). Two hours of restraint stress on the afternoon of

28

proestrus could equally block ovulation in adrenalectomized

and intact rats.

Control of Prolactin Secretion

 

While both suckling and cervical stimulation
illicits prolactin release, the dominant role of the hypo-
thalamus is inhibition of prolactin release. Severing
the pituitary stalk increase prolactin release (Donovan,
1970). When pituitaries were transplanted to the kidney
capsule gonadotropin secretion ceased and prolactin
secretion increased. Return of the pituitary from the
kidney capsule to the median eminence reestablished gonado-
tropin secretion, decreased prolactin release, and returned
ovarian cyclicity (Mess and Martini, 1968). Estrogen
enhances prolactin release while injections of testosterone,
progesterone, and in some cases cortisol and corticosterone
do not affect prolactin release (Meites, 1967). Reserpine,
and perphenazine which increased prolactin release, induces
pseudopregnancy in rats and reduces the amount of norepine-
phrine at the brain (Eisenberg, 1975; Chatterton, 1975).
The CNS dopaminergic system has been implicated as PIF
(prolactin inhibiting factor). Antidopaminergic drugs can
enhance prolactin secretion (Shaar, 1975). Prolactin is
markedly affected by various types of stress (Wakabayashi
et al., 1971; Wuttke et al., 1971; Ajika et al., 1972;
Linke et al., 1972; Euker et al., 1973). Ether stress and

blood sampling increased serum prolactin in male rats,

decrease<
afternOOJ
the mom:
a”, 197
restrain'
and cast:
decrease
(Euker e
Langlier
block et
r€leaSe
lactin,
OVarieC.
thetic .
is Cons
{Chatte

of PrOn

Flex-vQt
which
SawYe
he
inigl

ikLirc

29

decreased serum prolactin in proestrus rats and rats the
afternoon of estrus and had no effect on diestrus or rats
the morning of estrus (Riegle and Meites, 1976; Euker et
al., 1975; Bellinger and Mendel, 1975). Two hours of
restraint stress increased serum prolactin in diestrus,
and castrated female rats and intact male rats, and
decreased serum prolactin in proestrus and estrous rats
(Euker et al., 1973; Riegle and Meites, 1976). Harms,
Langlier, and McCann (1975) found dexamethasone could
block ether stress or disturbance stress induced prolactin
release while adrenalectomy augmented the release of pro-
lactin. Serum prolactin stabilized three days following
ovariectomy (Lawson and Gala, 1974) and stress by anes-
thetic or restraint increased prolactin release. Prolactin
is considered luteotrophic following ovulation in rats
(Chatterton, Chien, Ward, and Miller, 1975) and is capable
of promoting progesterone secretion.

Role of Catecholamines in Controlling
Hypothalamo-Hypophyseal Secretion

 

 

The presence of catecholamines in the central
nervous system was demonstrated in 1946 by von Euler,
Which he attributed to brain vasomotor nerves. In 1950
Sawyer suggested that catecholamines in the brain functioned
as neurotransmitters, or humoral transmitters to convey
information from the hypothalamus to the anterior pituitary

(Hiroshige and Abe, 1973). In 1970 Wurtman described six

possible

to regu.

variOus
hypothe
Par-ticI
to the
tropin
neurOt:
pituit,
releas.

releaSI

30

possible mechanisms of action brain catecholamines may use
to regulate hypothalamic secretion:

1. As neurotransmitters in a tract that provides an
input to hypophysiotrOpic cells in the hypothalamus.

2. As neurotransmitters that cause the hypothalamic
cells to secrete releasing hormones.

3. Act within the hypophysiotropic cell to release
releasing hormones.

4. Act as neurohormones directly on the anterior
pituitary.

5. Catecholamines delivered to the pituitary through
the general circulation and modify secretion of
releasing hormones, and

6. Catecholamines alter sensory input to the brain,
thus altering secretion of releasing hormones
secondarily.

Intrapituitary and intracerebral injections of
various catecholamines have supported or disputed Wurtman's
hypothesis. In recent years hypothalamic neurotransmitters,
particularly the catecholamines have been intimately linked
to the control of hypothalamic factors regulating gonado-
trOpin and prolactin secretion. However, the role of the
neurotransmitters on ACTH regulation is less clear. Intra—
pituitary injection of norepinephrine does not affect ACTH
release, while L-dopa and epinephrine could increase ACTH

release although not significantly (Hiroshige and Abe,

1973). 5
release 1
proved a
1969). 1
cholinerg
injection
carbachol
1974). '1
depletes
rhythms c
KaPlanski
l:
(1975) re
Stress de
the arena
tYIOsine
cateChOlé
totally E
REurOnS

31

1973). Serotonin and acctylcholinc had no ability to
release ACTH, while histamine released ACTH and spermidine
proved a potent releasing agent of ACTH (Pearce et al.,
1969). In 1972 Ganong hypothesized adrenergic inhibitory,
cholinergic stimulatory in CRF regulation. Intraventricular
injection of norepinephrine, dopamine, serotonin, and
carbachol increase serum corticosterone (Abe and Horoshige,
1974). Treatment with reserpine and 6-hydroxydopamine
depletes brain catecholamines but had no effect on circadian
rhythms or the stress response (Abe and Hiroshige, 1974;
Kaplanski, van Delft, Nyakas, Stoof, and Smelik, 1974).
Palkovits, Kobayashi, Kizer, Jacobowitz, and Kopen
(1975) reported that formalin injection, restraint and cold
stress decreased norepinephrine and dOpamine content of
the arcuate nucleus and repeated restraint stress increased
tyrosine hydroxylase at the arcuate nucleus which increases
catecholamine synthesis. The data presented does not
totally support Ganong's (1972) hypothesis that adrenergic
neurons in the hypothalamus are inhibitory for ACTH
release as depletion of norepinephrine or dopamine did not
affect ACTH release. While catecholamines may actually
increase plasma corticosterone (Abe and Hiroshige, 1974),
depletion of brain catecholamines by reserpine or 6-
hydroxydopamine or elevation of brain catecholamines had

no effect on circadian rhythms of ACTH or corticosterone,

or stra:

(Fuxe a]

32

or stress induced increase in CRF, and plasma corticosterone

(Fuxe and Hokfelt, 1969; Abe and Hiroshige, 1974).

Altamont
raised v
at Michj
temperat
of three
lights C
a COHURer

Water ad

rThis age
IJ‘BrfOrma
determin
in this

consiSte

anesthes

MATERIALS AND METHODS

Experimental Animals

 

All of the Long-Evans rats (Blue-Spruce Farms,
Altamont, N.Y.) used in these experiments were bred and
raised within the colony at the Endocrine Research Unit
at Michigan State University. The rats were housed in a
temperature controlled (22°C i 1°C) environment, in groups
of three or four, with a 12:12 1ight:dark schedule, having
lights on at 6 a.m. and lights off at 6 p.m. They were fed
a commercial laboratory rat diet (Wayne Lab Blox) and
water ad libitum.

The age of the rats used ranged from 3 to 5 months.
This age group is considered to be the peak of reproductive
performance. Vaginal cytology was monitored daily to
determine the reproductive status of the female rats used
in this study. Female rats were only used if they showed

consistent and regular 4 or 5 day estrous cycles.

Blood Collection

 

All blood samples were taken under light ether
anesthesia via suborbital sinus puncture within 40-60

seconds of initial cage disturbance. This procedure has

33

34

previously been shown to minimize the effects of stress

on blood hormone concentrations (Euker et al., 1975).
Between 1.0 and 1.5 m1 of blood was collected from female
rats and 1.5 to 2.0 m1 from male rats. The blood was
allowed to clot at room temperature for 30 to 45 minutes,
refrigerated at 4°C overnight and then centrifuged for 10
minutes at 3000 rpm in a refrigerated centrifuge (Sorvall
RC 2-B or RC-S). The serum was removed, frozen and stored

until assayed for prolactin, LH or testosterone.

Restraint Stress

 

Restraint stress was achieved by first lightly
anesthetizing the rats with ether and then securing them
in a supine position on a stainless steel laboratory table
by use of masking tape. Animals from the stress groups
were restrained in this fashion for two hours each morning

and afternoon for two weeks.

Weights
The rats were weighed before any experimentation
and at the end of the first and second week of the stress

regime.

Radio-Immunoassay for LH and Prolactin

 

All serum samples and pituitary homogenates were
assayed for LH and prolactin. LH and prolactin was

measured by double antibody radioimmunoassay as described

by MOI

perfox

1056 I
with l
was di
per 1C

well c

Niswen
Colora
with p
1:2800
was pr
dilUte«
Plexes
cific :
OVine a

CODCEnt

in Proc
Specifi
Pituita
dilutio;
mated f]

samples

35

by Monroe et a1. (1968) and Niswender et a1. (1969) and

performed routinely in the laboratory of Dr. J. Meites.
Purified rat prolactin (H—lO-lO-B-PRL) and LH (LER

1056 LH) were radioiodinated at Dr. J. Meites laboratory

with I125

on a 1 x 15 cm Bio-Gel P-60 column. The elutant

was diluted to a concentration of 30,000 counts per minute

per 100 pl with 1% gel PBS as counted on an automatic gamma
well counter (Nuclear Chicago, Des Plaines, Ill).

Antiovine LH antiserum was supplied by Dr. G.
Niswender (Colorado State University, Fort Collins,
Colorado). It was prepared by immunization of rabbits
with pituitary ovine LH hormone and was diluted to a
1:28000 concentration. The antirat prolactin antiserum
was prepared similarly using purified prolactin and was
diluted to a 1:5000 concentration. Antigen-antibody com-
plexes were precipitated using sheep antiserum from spe-
cific immunization against rabbit gamma globulin. This
ovine anti-rabbit gamma globulin was used at an effective
concentration which ranged from 1:35 to 1:60 dilution.

LH and prolactin radioimmunoassays are identical
in procedure and differ only in the labeled hormone and
Specific rabbit antisera. Duplicate aliquots of the
pituitary homogenate and incubation media were run at two
dilution volumes. Appropriate serum aliquots were esti—

mated from preliminary data. In all cases duplicate

samples were assayed if sufficient serum was available.

36

All unknown samples were placed in 12 x 75 mm dis-
posable culture tubes (diSPo(:2 Scientific Products,
McGraw-Park, Ill.) and made up to a volume of 0.5 ml using
1% gel PBS. Two hundred ul of either LH or prolactin anti-
sera was added, the tubes were vortexed and allowed to
equilibrate for 24 hours at 4°C. Then 100 pl of the
appropriate radioiodinated hormone was added, mixed and
allowed to equilibrate at 4°C for 24 hours. A final incu-
bation of 72 hours at 4°C followed addition of 200 ul of
the ovine anti-rat gamma globulin, which precipitates the
antigen-antibody complex. All the tubes then received 3
ml of cold 1% gel PBS and were centrifuged for 20 minutes
at 22500 x g. The supernatant was poured off and the tubes
allowed to drain upside down for 5 to 10 minutes. Care
was taken to keep the tubes chilled at all times during
this procedure to prevent the precipitate from sliding down
the tube wall. The tubes were then counted in the auto-
matic gamma counter.

Included in each assay were the reference standards,
total count tubes, total antibody binding tubes and non-
specific binding tubes. Total count tubes received only
100 pl of the labeled hormone and represented the radio-
active count recovery. Nonspecific binding was measured
by using 200 pl of rabbit serum (NRS) diluted with 1% gel
PBS in place of specific hormone antibody. Total antibody

binding was determined by using 1% gel PBS instead of

unknOi
15 di:
NIH-Ll
antibi
count
speci
from

Stand
Plott
stand
were

then

37

unknown serum. Reference standards consisted of 5 sets of
15 different quantities of purified NIH rat PRL-RP—l or
NIH-LH—RP-l. Counting time was calculated so that total
antibody binding minus nonspecific binding equalled 10,000
counts. Background counts were determined by the non-
specific binding tubes and was automatically subtracted
from the unknown tube counts by the gamma counter.
Standard curves were drawn on 3 cycle semi-log paper
plotting counts per minute against the log reference
standard concentration. Counts from the unknown samples
were then translated into nanogram amounts of hormone and

then transformed into ng/ml.

Competitive Binding Assay for Testosterone

 

Serum testosterone was determined by a single
antibody competitive binding assay (Mongkonpunya, 1973,
Michigan State University, East Lansing, Michigan). Serum
aliquots of 100 ul were extracted with 2 ml Benzene:
Hexane (1:1) in 17 x 100 mm DiSPo culture tubes, mixed
for 30 seconds and frozen at 0°C for 1 hour. The solvent
portion was poured off from the frozen aqueous portion
into 12 x 75 mm disposable culture tubes and dryed down
under air. A 0.5 aliquot of antitestosterone (diluted
1:3000) was added and mixed. The mixture was allowed to
equilibrate at room temperature for 30 minutes, and then
100 pl tritium labeled testosterone (H3-2,4,7,8,-testosterone,

New England Nuclear) was added, mixed, and allowed to

equili
dextra
PBS) w
was mi
and th
refrigr
Indust:
was rel
countec
Nuclea]
Purific
CurVes‘
include

middle

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NOHSPEC
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Uncham
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38

equilibrate for 18 hours at 4°C. One ml of charcoal with
dextran (250 mg charcoal, 25 mg dextran, 100 m1 of 1% gel
PBS) was added to precipitate the unbound hormone. This
was mixed for 10 seconds and chilled on ice for 10 minutes
and then centrifuged at 3000 rpm for 10 minutes in a
refrigerated centrifuge. Using a repipet diluter (Lab
Industries, Berkeley, Calif.) 0.5 m1 of the supernatant
was removed and added to 10 m1 scintillation vials and
counted in a automatic beta counter for 4 minutes (Chicago,
Nuclear). Reference standards were prepared using a
purified bovine testosterone preparation. Three standard
curves, consisting of 10 different concentrations were
included in each 100 tube run, one at the beginning, the
middle and the end.

Blank tubes, total count tubes, recovery tubes
and the standard serum tubes were also included so that a
correction factor could be determined to compensate for
procedural loss at extraction and for background counts.
Nonspecific binding was determined by using 100 pl of a
pooled rat serum, which is extracted and dried. One
hundred microliters (100 pl) of 1% gel PBS buffer was
added instead of antibody and the remaining procedure was
unchanged. Blank tubes receiving buffer instead of serum
or reference standard were used to determine total anti-

body binding. Total counts were determined by adding 10 ul

39

of 4 labeled tritium testosterone to scintillation vials,
plus 10 ml of scintillation counting cocktail.

Extraction recovery tubes were made with 100 pl
pooled rat serum, and 10 ul of 2 labeled tritium testo-
sterone. These were extracted and the solvent phase
poured off into scintillation vials, dried down and 10 m1
of counting cocktail added and counted.

Log hormone concentration was plotted on three
cycle semilog paper against counts per minute. The counts
from the serum unknown were then translated to nanograms
and multiplied by the correction factor to get nanograms

per m1 serum.

Catecholamine Assay

 

Hypothalamic catecholamine was performed at Eli
Lily Company (Indianapolis, Indiana) in Dr. James Clemens'
laboratory under the direction of Dr. Carl Shaar. A
modification of the microfluorescent technique of Laverty
and Taylor (1968) was used to measure dopamine and
norepinephrine. Seven to nine hypothalamic extracts from
control and stress groups from male and female experimental
groups were used for norepinephrine quantification, while
ihypothalamic extracts from control and restraint animals
from male experimental groups were used for measurement
of dopamine. Individual frozen hypothalamic extracts were
thawed, and transferred to a beaker with 2 m1 of 1 M sodium

acetate buffer and 1 m1 1% disodium EDTA. Heat activated,

40

neutral grade 1 alumina (240 mg) were added and the mixture
swirled for 5 minutes. The supernatant was poured off and
discarded. The alumina-catecholamine mixture was applied
to a microcolumn containing 140 mg of alumina. The column
was washed 3 times with triple distilled water, and then
the catecholamines eluted with 5 m1 of 0.2 N acetic acid
into evaporating tubes. These were evaporated to dryness,
and the residue resuspended in 0.4 ml of triple distilled
water and catecholamine determination were made by acid
fluorescence using a system of reverse blanks and an
internal standard. Ten microliters (10 ul) of phosphate
buffer at pH 6.52, 10 ul of 0.02 N iodine and 50 ul of
alkaline sulfite solution were added to 100 ml of the
resuspended extract. After 5 minutes 30 ul of glacial
acetic acid were added to develop the fluorescence.
Norepinephrine fluorescence was read at excitation wave
length of 392 mu and emission of 490 mu. Dopamine
fluorescence was measured following 40 minutes of heating

at 100°C at 320 mu excitation and 380 mu emission.

Experimental Design

 

The experiment was designed so that endocrine
parameters could be monitored or challenged while the
rats were undergoing a stress regime. The design can be
divided into three general areas:

1. response to stress over a two week interval,

41

2. in yiyg response to LHRH challenge before and
after chronic stress,
3. in yitrg response to LHRH challenge after
chronic stress.
The design was basically the same for both male and female
groups. Animals were selected by age, weight, and for the
females, regularity of estrous cycles. This was to ensure
that regular cyclicity did exist prior to experimentation
and that change in cyclicity could be attributed to the
stress regime. Minimum weight for the males was 225 grams
and for the females was 195 grams. Minimum weights were
selected to ensure a greater homogeneity of size in the
groups and to ensure that no animal was too small to
undergo the repetitive blood sampling. Mean weights taken
before and during the stress regime helped monitor the
effect of stress on the rats.
Each experimental group was divided into two
sections, one acting as control animals and the other as

the stress group. Both groups were subjected to in vivo

 

LHRH challenge before and after the stress treatments,
weekly blood samples and the in Vitro LHRH challenge..
Stress was achieved by restraining the rats as previously

described.

42

Model of the experimental design is as follows:

1-2 weeks

before stress

Day 1
2
3

10
11
12
13

14

+4 *3 C;

II

III

 

Selection of rats by age, weight and
vaginal cytology. First in vivo LHRH
challenge.

First day of stress, blood sample from
controls and following 2 hours of stress.

Weigh rats

Blood samples before and following stress,
and from controls.

Weigh rats

Second in vivo LHRH challenge.

 

Blood samples following 2 hours of stress;
in Vitro LHRH challenge.

Definition of Terms

 

Chronic Restraint Group
male

female

first experimental group
second experimental group

third experimental group

43

In vivo LHRH Challenge

 

Before beginning the stress regime, all animals
from both control and stress group were challenged with an
intravenous (i.v.) injection of synthetic LHRH (Eli Lily
Inc., Indianapolis, Ind.). LHRH challenge occurred in the
afternoon for both male and female groups. Female rats
were subjected to the first LHRH challenge only if they
were in the diestrus stage of the estrous cycle.

Following a control blood sample either 100 or
500 ng LHRH was injected via an exposed jugular vein.
Serial blood samples were taken at 15, 30 and 60 minutes
or 15 and 45 minutes postinjection. Following injection
and bleedings rats were returned to their cages. The same
process was repeated on the afternoon of day 12 or 13 of

Stress.

In vitro LHRH Challenge

 

Following the final morning of stress animals from
both the stress and control groups were decapitated, and
their hypothalami and anterior pituitaries were removed.
Control rats were decapitated within 30 seconds of initial
cage disturbance, while stress rats were decapitated
(immediately following removal from the stress table. This
was done in a room adjoining the colony to minimize stress
effects on basal levels of pituitary hormone in the control
animals, since this type of disturbance has been shown to

alter serum concentrations of prolactin and LH in male rats.

44

Hypothalamic Extract Preparation

The top of the skull was removed and the brain was
teased from the cranial cavity. The "hypothalamic island"
was located via the optic nerve tract on the ventral sur-
face. Iris scissors were used to remove this island at a
depth of 2 mm, and weighed to the nearest half milligram.
The hypothalami were hand homogenized in a total of 0.4 m1
cold 0.4 N perchloric acid and then centrifuged for 20
minutes at 20,000 rpm in a refrigerated ultra—centrifuge
(Sorvall RCZ-B or RC-S). The supernatant was removed and
placed in 12 x 75 mm disposable culture tubes with 0.1 ml
EDTA, and then frozen and stored for catecholamine assay.
Anterior Pituitary Collection
and Incubation

The anterior pituitary (AP) was teased from the
base of the cranium and placed in a petri dish on a piece
of filter paper soaked with medium 199 (1.1% Medium 199,
Difco Laboratories, Detroit, Mich.). The bilobar AP was
bisected at the isthmus with a scalpel blade and each half
was placed in a 12 x 75 mm diSPo culture tube containing
2 ml of medium 199 that had been previously incubated in a
.Lab-line shaker water bath at 37°C with 5% CO2 in 02 at a
rate to maintain a pH of 7.25-7.35. The tubes were
returned to the water bath at the same condition for one
hour. At the end of one hour the incubation-media was

poured off and the last remaining drops wiped from the

45

tube. The odd number tubes, containing one half of each
AP from the stress and control rats, 2 ml of medium 199
was added, and acted as control or untreated AP half. The
even number tubes containing the remaining AP half were
treated with 5, 25, 50 or 100 ug LHRH and medium 199 to 2
ml. The tubes were returned to the water bath and incu-
bated for 4 hours. The media was removed and diluted 1:10
with phosphate buffered saline (PBS) with 1% gelatin. This
solution was assayed for prolactin and LH released.
Pituitary halves were weighed and homogenized in a total
of 1.0 m1 1% PBS and then further diluted to 0.5 ml PBS/
mg pituitary. This solution was prepared for LH and
prolactin assay by dilution 1:40 with 1% PBS.

Plasma LH, Prolactin and

Testosterone Response
to Stress

 

 

 

Blood samples were taken at intervals to monitor
plasma LH, prolactin, and testosterone concentration as
affected by chronic stress. Blood samples were taken before
and after stress on days 1, 7 and 13 of the treatment
regime. Control animals were bled only once on the same
days. Pre-injection samples from the in_yiyg LHRH chal-
ilenges were used as the control samples for days 1 and 13;

this was done to minimize stress to the control animals.

.. _. mam

 

46

Statistical Analysis

Pituitary content, pituitary release and serum
hormone concentration were analyzed for drug and stress
treatment by analysis of variance. A critical alpha
probability value of 0.05 was selected. In studies where
experimental factors were determined to be significant,
the differences among the means were tested by the least
significant difference test (LSD) (Sokal and Rohlf, 1969).

In some cases the student t test alone was used.

RESULTS

LH Data

In Vivo Pituitary LHRH Challenge

 

Two doses of LHRH, 100 ng and 500 ng, were given
to the first experimental male group (C.Rmcf -I). Blood
samples were taken before and 15, 30 and 60 minutes post-
injection. This experimental group was subjected to the
in yiyg LHRH challenge only on day 10 of the stress
regime. A dose response was observed as both stress and
control groups had a greater response to the 500 ng dose
of LHRH than to the 100 ng dose (Figure 1). Preinjection
value for the 100 ng control group was 43.51 i 17.86 ng
LH/ml serum and 11.96 t 7.09 ng LH/ml serum for the 500 ng
LHRH control group. Resting values for the restraint groups
were 29.75 i 23.41 and 7.09 i 2.10 ng LH/ml serum for the
100 ng and 500 ng LHRH doses respectively. Although the
restraint groups arithmetic means are less than those of
the control group, the differences were not significant.
The response to the 100 ng LHRH dose did not differ
significantly from the control to the restraint group.

Peak response occurred at 15 minutes postinjection at

47

48

 

Fig. 1.--Effects of 100 ng and 500 ng LHRH on serum LH in
young male rats--C.R.d'-I.

Mean serum LH concentrations expressed as ng/ml
and their standard errors appear on the ordinate
as a function of a time relative to injection of
100 or 500 ng LHRH. Blood samples were taken
before injection (0 time) and 15, 30, and 60
minutes after intravenous LHRH injection. Solid
circles represent nonstress control animals,
while open circles signify chronically restrained
rats. The broken line represents the 100 ng LHRH
dose and the solid line the 500 ng LHRH dose.

Serum LH ng/ml

175

150

125

.5
O
O

N
G

50

49

 

 

Minutes After LHRH

50

68.50 i 8.19 and 54.0 i 5.08 ng LH/ml serum for the 100 ng
LHRH control and stress groups. By 60 minutes both con-
trol and stress rats serum LH had returned to resting
levels. The response to the 500 ng LHRH dose did not
differ significantly at 15 minutes from control to stress
group, the control group having 92.88 i 43.69 ng LH/ml
serum and the restraint group 122.17 i 17.34 ng LH/ml serum.
The stress groups peak response was measured at the 15
minute bleeding while the control group peak response was
measured at 30 minutes at 185.44 i 58.77 ng LH/ml serum.
The value from the stress group at 30 minutes was 73.63 i
4.50 ng LH/ml serum which was significantly less than con-
trol value at the same time. By 60 minutes the restraint
group had returned to the equivalent of resting levels
while the control group still had elevated levels, 73.69 i
17.53 ng LH/ml serum.

Since the preliminary data suggested that there
were differences in stressed male rats response to 500 ng
LHRH the experiment was repeated in duplicate (C.R.CfV—II,
C.R.<5'-III, Figure 2). Blood samples were taken before
and 15 and 45 minutes postinjection. In both C.R.Cf«-II
.and C.R.<j'-III experimental groups stress had no effect
on the pituitaries ability to respond to a bolus injection
of LHRH. The prestress LHRH challenge response did not
differ significantly from control or stress response at

any of the time sampled. While the control rats resting

51

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much pommmuum mHHmOHcouno ucomoumou moaouflo ammo can mosam> mmouum

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levels of LH were 8.37 i 2.95 and 16.74 i 7.28 ng LH/ml
serum for C.R.Cf -II and C.R.cf-III respectively, the
stress groups resting levels were 3.33 i 0.0 and 4.09 i
0.39 ng LH/ml serum for the two groups. Although the
resting levels of the stress group were less than the con-
trols there was no significant difference. There was also
no significant difference in peak values measured at 15
minutes as controls peak values were 87.81 i 9.09 and
39.14 i 6.44 ng LH/ml serum for C.R. 0’ -II and C.R. 0' -III
and 90.17 i 8.84 and 82.40 i 8.27 ng LH/ml serum for the
two stress groups. The 45 minute postinjection serum
concentration also showed no alteration in the pituitaries
ability to respond to LHRH. In both control and stress
animals from both experimental groups serum LH was returning
towards resting levels. Although the resting serum LH
concentrations were suppressed by the chronic stress, the
pituitaries ability to respond to LHRH is unaltered.

Less consistent data came from the 2 female experi-
mental groups (Figure 3). Only the restraint animals from
the first group (C.R.9 -I) were challenged with LHRH,
once before the stress regime and then on day 13 of stress.
For the first LHRH challenge all of the rats were diestrus
day one of their estrous cycle, and were in constant
diestrus following chronic stress for the second LHRH
challenge. Resting serum LH concentrations were 26.10 t

3.47 ng LH/ml serum before the stress regime and had

54

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dropped significantly to 3.78 i 0.29 ng LH/ml serum after
13 days of stress. Peak values were measured with the 15
minute blood sample. The prestress peak value of LH was
312.60 i 18.40 ng LH/ml serum. Following 13 days of stress
the peak value was only 212.90 i 19.23 ng LH/ml serum,
which is significantly less than the prestress peak value.
Forty-five minutes postinjection the prestress values
decreased to 73.40 i 10.0 ng LH/ml serum.

In the second group, C.R. 9 -II, the LHRH challenge
gave somewhat differing results. The control, prestress
and stress groups resting serum LH concentrations did not
differ significantly. Resting LH levels for the control
group were 38.06 i 12.23 ng LH/ml serum and 21.01 i 6.66
ng LH/ml serum for the stress groups. Peak values of LH
following LHRH injection were measured at 15 minutes post-
injection in both control and stress groups. There was no
significant difference between peak values following 2
weeks of stress as controls had 237.78 r 23.26 ng LH/ml
serum and the stress group 219.68 1 16.43 mg LH/ml serum.
At 45 minutes postinjection the stress group had signifi-
cantly less LH than the control group. Control rats serum
LH values at 45 minutes were 120.34 i 39.61 ng LH/ml serum
while the stress groups peak value was 60.22 t 18.54 ng LH/
m1 serum. In both chronic restraint female groups, stress

decreased the pituitaries ability to release LH by LHRH

57

stimulation by either decreasing the maximal response or
duration of response.

In Vitro Pituitary LHRH
Challenge

The anterior pituitary halves from C.R.Cf -I were
incubated with either 0, 25 or 100 ng LHRH. Both of the
doses of LHRH stimulated release of LH maximally, so in
C.R.O"I -II and C.R. 0" -III 5 and 50 ng of LHRH were used
to incubate the anterior pituitaries.

Pituitary content of LH was not affected by LHRH
or stress treatment. Control animals from C.R.cf —I
without LHRH treatment had 84.15 i 7.70 ug LH/mg pituitary
tissue, pituitaries treated with 25 ng LHRH contained
90.20 i 4.54 ug LH/mg pituitary and pituitaries treated
with 100 ng LHRH had 81.55 i 4.23 ug LH/mg pituitary
tissue (Figure 4). Stress animals had 85.67 i 2.82,
79.77 i 4.55 and 80.56 i 5.60 pg LH/mg pituitary after
treatment with 0, 25, and 100 ng LHRH respectively. While
pituitaries from CAR.CT -II and C.R.cf -III contained
approximately one half as much LH as rats from C.R.(5'-I,
they also showed the consistent LH content regardless of
_1LHRH dose or stress.

LH released from the anterior pituitary halves
.incubated with LHRH showed a dose response relationship
(Figure 5). Pituitary halves treated with 5 ng LHRH

:released significantly more LH than those receiving none.

58

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59

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62

LHRH doses of 25, 50 and 100 ng stimulated maximal amounts
of LH release, which was greater, but not significantly
than the amount released by 5 ng LHRH. Two weeks of
restraint stress had no apparent effect on pituitary
responsiveness to LHRH stimulation in male rats.

Chronic stress affected the pituitary content of LH
in female rats differently. LH content of pituitary halves
from control rats was unaffected by LHRH dose, as was the
content in pituitary halves from stressed rats; but pitui-
tary halves from stress rats contained significantly more
LH than pituitary halves from control rats (Figure 6).
Control rats pituitary content ranged from 5-9 ug LH/mg
pituitary while pituitary content of stressed rats ranged
from 12-18 ug LH/mg pituitary tissue.

LH release from pituitary halves also showed the
stepwise increase release of LH with increasing LHRH dose
stimulation (Figure 7). Stress had no effect on the amount
of LH released in response to LHRH as control values and

stress values are similar for each dose of LHRH.

Stress Effects on Serum LH

 

Blood samples were taken once a week from both the
control and restraint stress animals from all experimental
groups to monitor stress effects on resting serum LH con-
centrations. The effect of stress on serum LH on day one
of treatment was variable (Figure 8). In both C.R.C7'-I

and C.R.c7°-III serum LH dropped significantly following

63

.msonm Como CH maoEHCo mo HoQECC oCu onoHUCH mung oCu mo omoa
oCu um mquECz .>Ho>Hpoommou 0C om UCo m mo momoc mama ouooHUCH mnoa
oopoam UCo mama woCHH oHHC3 .meoEumouu mama 0C mCH>HoooH moHHouHCuHa
uComouaou mama Como .aaaa CpH3 uCoEuoonu mCHsoaaom Coos on» no uonuo

pumchvm oau ou mCHCCoamoHHoo muoxooun Cqu AoCmmHu muouHCuHm mE\aa may
muouHCuHm oCC CH COHuoHuCoOCoo ma Coos muComoumoH Hon Como mo uanoC one

.muou oaoEom mCCom CH ma mo uCouCOO muouHCUHa Co mama mo uoommo oCBII.o

.mHm

64

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

13
O

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

‘5 . IIIIIIHHHHH((((iZ
H a. o

 

20
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O

 

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Stress

Control

Stress

Control

65

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m nuHB mouoouu mo>aon auouHsuHm Eoum omooaou ma onooHUCH muon momonm
mCo muon moCHH oHHn3 .unofiuoouu mama 0C mCH>HoooH mo>amn muopHCuHm
uComoHQoH muon Como .mama nuH3 uCoEuooHu Houmo ma «0 omooaou anon
IHCuHm muComoumoH Hon nomm .CooE on» mo uouuo muomCoum onu op mCHUCoa
Imouuoo muonooun nuH3 AoCmmHu muonHCuHm mE\ma may mo>aon muopHCuHm
Comm momooaou CoHuouuCooCoo ma Coos muComonou Hon nooo mo uanon one

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66

 

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rat.— on

 

 

 

 

 

 

 

mac. 5

 

 

 

 

 

 

 

Ta. .3

 

6w /6d escalaa H 1

67

 

Fig. 8.—-The effect on chronic restraint stress on serum LH
in young male rats.

The height of each bar represents mean serum LH
concentration (ng LH/ml) with brackets corres-
ponding to the standard error of the mean.

Each bar represents serum LH on different days of
stress treatment. Open bars represent control
animals resting serum LH levels, lined bars
represent restraint rats resting serum LH con-
centrations, and shaded bars represent restraint
rats serum LH concentrations following 2 hours
of restraint stress. Asterisk marks serum
samples having too little LH concentration to be
measured by the assay so there is no standard
error of the mean.

Serum LH ng/ml

C

C

     

C

38!”: as 2!:
Day 1

RS on as an
Day 1

RSOII RS 2!!
Day 1

68

C 38!": RS 2!:
Day 5

C.R. d'-n

C 880'! RS 2h

Day 7

C.R. J—m

C RS 0!! RS 2h
Day 7

C

C

C

 

RSOh 352»
Day 13

 

RSOh as 2!:
Day 14

350'! RS 2!!
Day 13

69

two hours of restraint stress from 35.89 i 5.95 to 21.40 i
2.79 ng LH/ml serum and 22.07 i 7.08 to 6.61 i 1.08 ng LH/
ml serum respectively, while C.R.Cfi-II demonstrated a
significant increase in serum LH following two hours of
stress. After one week of stress resting levels of LH had
decreased from 35.89 i 5.95 to 14.19 i 2.72 ng LH/ml serum
for C.R.t77-I, from 21.77 i 3.40 to 3.63 i 2.10 ng LH/mg
serum for C.R.:7’-II and 13.35 i 3.7 to 9.52 i 3.29 ng LH/

mg serum for C.Rac7’-III. Following two hours of restraint

 

serum LH decreased from the already lowered resting levels
to the minimum sensitivity of the LH assay. Results from
the end of the second week were similar for C.R.C7t-II

and C.R.<7’-III where resting serum LH levels from the
restraint groups were less than those after controls and
following two hours of restraint serum LH concentrations
remained lowered. Although C.R.Cf'-I resting LH concen-
tration of the restraint group is not different from the
control, two hours of stress decreased serum LH.

Changes in serum LH in response to stress in female
rats were found to be dependent upon the stage of the
estrous cycle at the time of stress (Figure 9). Restraint
stress on diestrus either decreased serum LH as in C.R.g) -II
‘where serum LH decreased from 54.17 i 9.63 to 25.13 i 4.64
or had no effect as with C.R. g -I where serum LH was
9.48 i 3.06 before stress and 11.62 i 3.73 ng LH/ml

following stress. Restraint stress on the afternoon of

 

Fig.

70

9.--The effects of stress on serum LH in young female

rats.

The height of the bar represents mean serum LH
concentration expressed as ng LH per m1 serum
with brackets corresponding to the standard error
of the mean. Each bar represents serum LH on
different days of stress treatment. Open bars
represent control animals resting serum LH
levels, lined bars represent restraint rats (RS)
resting LH levels, and shaded bars represent
restraint rats serum LH levels following 2

hours of restraint stress. Asterisks mark groups
with no standard error of the mean due to serum
samples containing too little LH to be measured.

Serum LH ng/ ml

1151

75

50

25

. I

 

 

 

a " ‘

 

   

0h 2h 0h 2h Oh 2h 0h 2h Oh 2h
Diestrus Proestrus Estrus Diestrus Diestrus
Day 1 Day 6 Day 13

C.R. Q-n

 

C RSOhRS2h C RSOhR82h C RSOhRSZh
Day 1 Day 7 Day 14

72

proestrus decreased serum LH from a proestrus peak of
114.47 i 18.83 ng LH/ml serum to 76.89 i 26.35 ng LH/ml
serum. Restraint stress during estrus appeared to have no
significant effect on serum LH concentration. After one
week of stress, resting serum LH was decreased to the
minimum sensitivity of LH assay and two hours of stress
did not change serum LH. Resting LH concentrations were
also at a minimum following two weeks of stress for

C.R.? -I and two hours of stress did not alter serum LH.
Resting serum LH from the stress animals of C.R.S? -II was
not significantly different from control resting levels at
the end of the second week as it was 26.57 i 8.28 ng LH/ml
serum while the control value was 38.06 i 12.25 ng LH/ml
serum. Two hours of restraint stress did decrease serum

LH to the lowest sensitivity of the LH assay.
PRL Data

In Vitro Pituitary LHRH
Challenge

 

 

Pituitary prolactin content of male rats was not
affected by LHRH stimulation and was not altered by two
weeks of chronic stress (Figure 10). The pituitaries con-
tained between 4.6 and 6.0 ng PRL/mg pituitary. Prolactin
release was also unaffected by LHRH stimulation or by

restraint stress.

73

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anz mouoouu moHHMUHCnHa ouooHUCH muon mononm UCo mama 0C mm ans nouoouu

mo>aon auonHCuHm uComonoH muon UoCHH .uCoEuooHu mama usonuH3 moHHou
IHCuHa uComouaon mumn Como .CooE onu mo uouuo nuocCoum onu on mCHUCoa
Imouuoo muonooun nqu .oCmmHu muouHCuHm ma mom CHuooHoum a: no omooHoH
aumnHCuHm Coos nCo uCouCoo muouHCuHa Coos muComoHaoH won on» mo uanon one

.muou oHoE mCCom CH CHuooaoum omooaou
on auHHHno UCo CHuomaoum mo uCouCoo auouHsuHm Co mmouum mo muoommo oneun.oa .mHa

 

 

74

moonuw

 

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IE3 ac

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9)
Stu/err ugmenwd

75

Stress Effects on Serum
Prolactin

 

 

Serum prolactin was relatively unchanged through-
out the two week stress in male rats (Figure 11). Resting
levels of prolactin in stress and control rats were
similar and two hours of restraint stress had no signifi-
cant effect on day l of stress in C.R.O" -I or C.R.d -III.
At the end of the first and second week of stress, serum
prolactin concentrations was not significantly different

from control to stress rats. Two hours of restraint

 

stress did not alter serum prolactin from the resting

values.

Testosterone Data

 

Serum Testosterone Response
to LHRH Challenge

 

 

LHRH significantly elevated serum testosterone
over the 45 minute interval sampled in the control animals
from 1.55 i 0.34 ng testosterone/m1 serum preinjection to
3.0 i 0.44 ng testosterone/ml serum at 15 minutes and
4.10 i 0.56 ng testosterone/ml serum (Figure 12). Follow-
ing two weeks of stress resting levels of testosterone
were suppressed to 0.56 i 0.05 ng testosterone/ml serum.
Injection of 500 ng LHRH tended to increase serum testo-
sterone but not significantly to 0.65 t 0.08 ng testo-
sterone/m1 serum at 15 minutes and 0.70 i 0.10 ng testo-

sterone/m1 serum at 45 minutes postinjection.

 

Fig.

76

11.--The effect of stress on serum prolactin in younqgr

male rats.

The height of the bar indicates mean serum pro-
lactin concentration expressed as ng prolactin
per ml serum, with brackets corresponding to thtEB
standard error of the mean. Each bar representgss
serum prolactin on different days of stress
treatment. Open bars represent control animals
resting serum prolactin, lined bars represent
restraint rats resting levels, and shaded bars
represent rats serum prolactin levels following

2 hours of restraint stress. Numbers at the

base of the bars represents the number of

animals in each group.

ng/ml

Serum Prolactin

77

   

C RSOhRSZh C RSOhRSZh C RSOhRSZh
Day1 Days Day 12

5O

 

C RSOhRSZh C RSOhRSZh C RSOhRS 2h
Day 1 Day 7 Day 13

 

78

Fig. 12.--The effects of 500 ng LHRH on serum testosterone

in young male rats.

Mean serum testosterone values (ng/ml) and their
standard errors of the mean appear on the
ordinate as a function of time. Blood samples
were taken before injection (0 time) and 15

and 45 minutes postinjection with 500 ng LHRH.
Solid circles represent control animal values,
while open circles represent serum testosterone
values from chronically restrained rats. Numbers
indicate number of animals per group.

Serum Testosterone ng/ml

79

I

/

 

i 9 3

4
2
C
l
2.,
0*
0

Minutes After LHRH

 

80

Effect of Stress on Serum
Testosterone

Serum testosterone from control animals taken at
one week intervals remained unchanged, ranging between
1.00 to 2.00 ng testosterone/ml serum (Figure 13). The
first two hour restraint period had no significant effect
on serum testosterone. After one week of stress resting
testosterone concentrations in the stress group was sig-
nificantly decreased to 0.36 i 0.03 ng testosterone/m1
serum. Further stress did not change serum testosterone.

Serum testosterone was similarly suppressed at the end of

two weeks of stress.

Catecholamine Data
Hypothalamic extracts from control and stress male
and female rats were assayed for dopamine and norepinephrine
content (Table 1). Chronic stress had no effect on hypo-
thalamic content of norepinephrine in male and female

.rats. Dopamine content was also unaffected by restraint

stress in male rats.

Vaginal Cytology Data
Regular estrous cyclicity was part of the criteria
le'the selection of female rats. Restraint stress inter-~
rupted the estrous cycles in all of the female rats that
underwent the two week stress treatment. By the morning
of day 4 of stress vaginal cytology of the restraint female

groups were of the leukocytic cell type, which is

 

81

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mcflumou mumn ucflmuumou uaommumwu mums pmcfla .mmSHm> oconmumoummu Edumm
mcflummu mHmEHcm Honucoo ucwmmnmou mumn some .ucwaammuu mmmuum no

what ucmuwMMflp :0 mcoumumODmmu Esumm mwcmmonmmu Hon zoom .cmmE may mo
Houuo pumocmum may on Unaccommmuuoo mumxomun nuflz Edumm HE\o:oumumovmmu
ma mm mcoumpmoumou Eduwm some mucmmmummu mumn may no wzmfion one

.mumu mama masow CH ocououmoummu Eamon co mmmuum mo powmwo oneuu.ma

 

.mam

82

 

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84

representative of the diestrus state. All rats remained
diestrus throughout the two week interval except for 2 of
16 rats from C.R.g? -I that had proestrus smears on day 8
of stress, and 5 of the 13 rats from C.R.S} -II were
proestrus or estrus on day 8. On day 8 of stress the
restraint rats were bled twice and this may have caused the
change from diestrus cytology. The control groups for

each stress group continued to cycle.

Chronic Stress Effects on Body Weights

 

 

Two weeks of restraint stress caused a significant
decrease in body weight in female rats from 230.21 1 6.74 g
to 214.08 3 5.96 9 (Table 2). Over the same two week span
the control females had a significant increase in body
weight from 244.40 1 8.82 g to 268.0 i 7.69 g.

The control male groups also showed no loss of
body weight (Table 2). C.R.(jz—I controls weighed 487.07 i
18.7 g at the beginning of the experiment and 465.77 1
15.14 g at the end. C.R. O"-II and C.R. 0" -III showed
similar results. The chronically restrained male rats
had a significant decrease in body weight as C.R.(7'-I
dropped from 453.38 1 11.31 g to 399.0 i 10.02 g, while
C.R.(ja-II had a decrease from 478.0 t 20.54 g to 413 :

16.99 g and C.R.Oa-III 496.5 i 21.20 g to 423.0 i 20.07 g.

85

Table 2.--The Effects of Chronic Restraint Stress on Body Weight of
Male and Female Rats.

. -.--—--...—--- -77..--fia - W —.
_—.——___....-~.——— -7 _-——-—_—-—— . - .. —..._. —.~ .— a-..-- ~-

 

No. Body Weight (grams)
C.R. (IT-I Day 1 Day 5 Day 12
Control 13 487.07 i 18.70 481.15 i 15.97 465.77 i 15.41

Restraint 26 453.3 f 11.31 429.62 i 10.83* 399.0 f 10.02*
C.R. Cf—II Day 1 Day 6 Day 14

Control 10 472.03 i 9.75 461.0 i 13.47 456.25 i 21.03

Restraint 10 478.0 i 20.54 427.7 i 19.65* 413.33 i 6.99*
C.R.<3'-III Day 1 Day 8 Day 14

Control 7 444.29 i 17.30 445.0 i 14.23 465.0 1 12.04

H-

Restraint 10 496.5 i 21.20 445 20.62* 423 i 20.09*

C.R. 9 -II Day 1 Day 13
Control 10 244.4 i 8.82 268 t 7.69**
Restraint 14 230.21 i 6.74 214.08 i 5.96*

 

Weights are shown as mean 1 SEM in grams.

*Denotes a significant decrease in body weight from day 1
values.

**Denotes a significant increase in body weight from day 1
values.

 

DISCUSSION

The results indicate that chronic restraint stress
can effectively interfere with reproduction in both male
and female rats. Stressed female rats did not have regular
estrous cycles, stressed male rats had lowered serum
testosterone, and serum LH was suppressed in both female
and male rats. Stress has been shown to interfere with
reproduction in rats. Restraint stress during pregnancy
has been found to increase embryonic mortality in the rat
(Euker and Riegle, 1973). Plasma corticosterone increases
during pregnancy in the hamster, but 24 hours of restraint
stress, or isolation without food, or injection of corti-
costerone on day 14-15 of gestation increases cortico-
ster-ne levels even more and significantly increases the
incidence of cleft palate in the offspring (Barlow, Mc-
Elhalton, Morrison and Sullivan, 1974). Heat stress has
been found to impair reproduction in ewes (Neville and
Neathery, 1974) and increase infant mortality and decrease
weaning weight in rats and hamsters (Hearnshaw, Wodzicka-
Tomaszewska, 1973; Barlow, Morrison and Sullivan, 1975).
Stress in the form of handling, ether or serial blood

samples increases serum LH in male rats (Euker, Meites and

86

87

Riegle, 1975). Two hours of restraint stress had no effect
on serum LH in male, diestrus, or estrus rats and decreased
serum LH in proestrus rats (Euker, Meites and Riegle, 1973).
Resting levels of LH were affected by chronic
stress. The response of male rats to the initial stress
was variable as both elevation and suppression of serum LH
occurred, which correlates with the ether stress data and
restraint stress data of Euker et al. (1973, 1975). These
data suggest that the intensity of the stress can affect
serum LH levels in the male rat. Restraint stress may be
considered more stressful than either ether stress or
handling, as these are carried out over a very short time,
generally not exceeding 15 minutes, while restraint stress
was carried out for 2 hours. It is possible that restraint
stress initially elevated serum LH, but after 2 hours the
serum LH was returning to resting values or had returned
to resting values, or was suppressed by the sustained
stress. Following 1 or 2 weeks of stress most male rats
did experience a decrease in resting serum LH and in many
instances two hours of stress could further decrease serum
LH. In some rats serum LH appeared unaffected by the
chronic stress as resting LH levels following a week of
stress were not significantly different from control
animal resting levels. This suggests that serum LH

secretion could recover from stress-induced suppression in

 

 

88

male rats and that stress may have only a temporary effect
on LH secretion.

In comparison to the male rat the female rat
appears to be more sensitive to stress-induced inhibition
of LH secretion. Restraint stress caused complete suppres-
sion of resting levels of serum LH far below serum LH con-
centrations found in estrous or diestrous female rats or
chronically stressed male rats. Where two hours of restraint

stress could further decrease serum LH in male rats it could

 

not decrease serum LH in female rats. Resting serum LH
concentrations in female rats were found to be at the lower
limits of the LH assay sensitivity, which suggests that
chronic stress can completely suppress LH release in female
rats where it partially or temporarily decreased serum LH
in male rats. Resting serum LH blood samples were taken
four hours following the morning restraint period. Although
the suppressed serum LH following two hours of restraint

in male rats returned toward normal resting levels four
hours post stress, female rats serum LH remained maximally
suppressed four hours following completion of the stress
regime.

Pituitary responsiveness was tested by measuring
the increase in serum LH stimulated by an intravenous
injection of 100 or 500 ng LHRH. If stress was exerting
an effect at the pituitary, we would expect pituitary

responsiveness to LHRH to be affected. No significant

89

difference was observed between the control or stress rats
receiving 100 ng LHRH over the sampling times used in this
study. Stress and control animals receiving 500 ng LHRH
released more LH at peak response than the rats receiving
the 100 ng LHRH dose. In C.R. 0" -II and C.R. O'-III there
was no difference in LH response to LHRH between stress and
control male rats. C.R.Cf'-I rats achieved a peak value at
30 minutes for control rats, while the stress group had
reached peak serum LH secretion at 15 minutes and by 30
minutes was returning towards the resting concentrations.
Although preinjection serum LH concentrations were less in
the stressed rats, peak values measured at 15 minutes and
duration of LH release were similar, except for the one
control group mentioned. These three experiments suggest
that the pituitary of the stressed male rat retains its
responsiveness to LHRH, implying that stress may be
affecting LHRH availability to the pituitary.

A bolus injection of 500 ng LHRH was also used to
determine chronic stress effects on the control of LH
release in female rats. Resting serum LH was suppressed
by the chronic stress in female rats and exogenous LHRH
was used to help determine if stress exerted its effects at
the hypothalamus or the pituitary. The first restraint
group, C.R.S? -I, released 31.90% less LH in response to
LHRH following 2 weeks of restraint stress than they did

before the stress. Peak release was measured at the 15

 

90

minute sample, and by 45 minutes postinjection both groups
were returning towards the preinjection LH values while
peak response following injection with 500 ng LHRH for the
stress group C.R.ED -II was not significantly different
from the control groups value, overall secretion may have
been less. By 45 minutes postinjection stress rats serum
LH concentration had decreased to half of the control serum
LH levels, which suggests a possible decrease in pituitary
responsiveness in female rats following two weeks of chronic
restraint.

The in yitrg LHRH response of male rats supported
the in zizg LH data from C.R.CT -II and C.R.C7 -III.
Chronic stress had no effect on LH content or release of
LH from the pituitaries of control and LHRH stimulated
pituitaries. The amount of LH released from the pituitary
was related to the dose of LHRH used and not to the stress.
From the in yiyg and in XEEEQ LHRH data from C.R.CT-II
and C.R.CT -III, it can be hypothesized that chronic
restraint stress does not effect the ability of the pitui-
tary to synthesize or secrete LH in male rats. The effects
of stress to lower resting serum LH concentrations in male
rats does not appear to be controlled by a change at the
pituitary, but at higher centers. Stress can alter serum
glucocorticoids and adrenal catecholamines (Scheiber, 1963;
Mountcastle, 1974) which can feed back at the pituitary

and hypothalamus directly or indirectly and can alter LH

 

 

91

and prolactin secretions (Swerdloff, Jacobs and Odell,
1972). Injection of hydrocortisone, prednisolone or ACTH
during follicular development blocked the estrogen rise
and delayed ovulation in sows (Liptrap, 1970) which indi-
cates interference in LH secretion. In 1972 Ganong sug-
gested that catecholamines may regulate hypothalamic
function but conflicting data as to catecholamines stimu-
lating or inhibiting ability has not been elucidated
(Hiroshige and Abe, 1973, 1974; Kaplanski, VanDelft,
Nyakas, Stoof and Smelik, 1974; Eisenberg, 1975; Palkovits,
1975). It has also been shown that stress can change
serum LH directly in adrenalectomized rats, who have lowered
levels of adrenal glucocorticoids or adrenal catecholamines
(McKay, Wood and Riegle, 1976). Thus stress may also be
mediated in some manner at higher centers, by changing
neural input at the brain or by changing the sensitivity
of the hypothalamus to LH or testosterone negative feedback.
LHRH treatment stimulated similar LH release from
pituitary halves of the control and stressed female rats.
However pituitary LH content was higher in stressed com-
pared to control groups. Although these studies show that
the primary effect of stress on gonadotropin control mech—
anisms is at the level of the hypothalamus, the increase
in pituitary LH content suggests that stress may also be
affecting LH synthesis and release at the level of the

pituitary. Since LH release following LHRH stimulation was

 

 

92

similar in control and treated groups, the increase LH
content may be caused by storage granules not available
for secretion. These experiments do not indicate whether
stress effects on pituitary LH content are mediated by
unidentified hypothalamic factors or other neural factors
or alterations in adrenal function affect the pituitary
directly. While the data suggests that the hypothalamus
or some higher center and not the pituitary is changed

by chronic stress in male rats, it also suggests that both

 

the pituitary and higher centers are affected by stress in
female rats.

Prolactin is a hormone that is also affected by
stress. Acute stress such as new environment, ether
stress or handling results in an increase in serum pro-
lactin in male rats (Euker, Meites and Riegle, 1975;
Seggie and Brown, 1975; Brown, Uhler, Seggie, Schally and
Kastin, 1974). Two hours of restraint stress increased
serum prolactin in male rats (Euker, Meites and Riegle,
1973; Riegle and Meites, 1976). In the chronic restraint
experiments two hours of restraint gave mixed results.
C.R.Cf'-I exhibited the previously established increase in
serum prolactin, while two hours of restraint for
C.R.Cf -III serum prolactin either decreased or remained
unchanged from control values. It is possible that the
C.R,<5'-III stress group serum prolactin did initially rise

in response to the stress, but had begun to return towards

 

93

nonstressed control values. This suggests that there is a
limit to the stress induced release of prolactin. Following
the first and second week of stress serum prolactin was not
significantly affected by two hours of restraint. Treatment
with dexamethasone is known to suppress stress-induced CRF
and ACTH release (Harms, Langlier and McCan, 1975; Euker,
Meites and Riegle, 1975), and can block stress-induced
prolactin release caused by distrubance (Harms, Langher
and McCann, 1975) or serial bleeding (Euker, Meites and
Riegle, 1975). It is possible that stress-induced alter-
ations of adrenal glucocorticoid secretion can influence
pituitary prolactin release. Apparently the animals expe-
riencing chronic stress adapt to the procedure, or high
stress-induced levels of glucorticoids may have inhibited
hypothalamic-hypophyseal mechanisms allowing increased
prolactin release.

It was not surprising to find chronic stress had
no effect on pituitary content or release of prolactin, as
serum prolactin was unchanged with chronic stress. Pro-
lactin release from pituitary halves from stress and con-
trol animals were not significantly different nor was
pituitary content of prolactin, which suggests that chronic
stress had no effect on prolactin release at the pituitary.

The data from both the in vivo and in vitro experiments

 

indicate that chronic stress does not alter prolactin

release from the pituitary and agrees with the hypothesis

 

 

94

of Harms et a1. (1975). The data from these experiments
do not indicate whether hypothalamic factors that inhibit
prolactin secretion are changed by chronic stress.

The unique distribution of brain biogenic amines
in the hypothalamus suggests an important role of catechol-
amines in the neuroendocrine regulation of anterior pitui-
tary ACTH secretion (Fuxe and Hokfelt, 1969). The
adrenegic system was hypothesized to inhibit pituitary
secretion and cholenergic as stimulatory (Ganong, 1972).
While Abe and Hiroshige (1973, 1974) found catecholamines
increased pituitary release. Catecholamines, especially
dOpamine can inhibit release of pituitary prolactin by
affecting hypothalamic release of factors inhibiting pro-
lactin secretion or by direct hypophyseal inhibitors (Shaar
and Clemens, 1974). Hypothalamic content of dopamine and
norepinephrine were found unchanged in chronically stressed
rats when compared to control rats. This data suggests
that hypothalamic catecholamines are unaffected by chronic
stress which is reflected in unchanged pituitary content
and serum concentrations of prolactin with chronic restraint
stress. Although the whole hypothalamic content of
catecholamines appears to be unaltered by stress, it may
not be the best way to measure the effect of chronic stress
on brain catecholamines. Whole hypothalamic catecholamine
content may not be as important as specific loci within the

hypothalamus, as both acute and repeated stress was found

95

to decrease norepinephrine and dopamine content in the
arcuate nucleus without changing catecholamine content in
the rest of the hypothalamus (Palkovits et al., 1975).
Since the arcuate nucleus is only 5% of the hypothalamic
weight, measurement of whole hypothalamic catecholamines
may fail to detect alteration in such a small area. A
change in turnover of catecholamines may also be important
as a stress effect, as the enzyme tyrosine hydroxylase

used in aminergic synthesis increases in the arcuate

 

nucleus with stress (Palkovits et al., 1975). This sug-
gests that norepinephrine and/or dopamine are being syn-
thesized and secreted at an increased rate with stress.
Studies measuring catecholamine turnover within areas of
the hypothalamus may be beneficial in understanding hypo-
thalamic catecholamines role in stress on gonadotropin and
prolactin regulation.

Serum testosterone was measured in male rats and
daily vaginal cytology monitored in the female rats as an
indication of reproductive capabilities. While restraint
stress had no effect on serum testosterone on day l of
stress by day 7 resting levels of testosterone before and
after stress treatment were less than one-third of control
animal values. Although resting serum LH levels of
stressed groups were not significantly different from serum
LH concentrations in control rats, the decrease in serum

testosterone levels indicate decreased gonadotropin

 

96

stimulation of the testes. Serum LH was lowered during the
twice daily stress periods. Perhaps this interfered with
the release of LH necessary for testosterone synthesis and
release, as the pulsatile release of LH was blocked by
immobilization stress (Blake, 1975). While stress appears
to have a direct effect at the testes as serum LH increased
following LHRH injection and testosterone did not, it is
more likely that the testes did not respond because it had
been inactive for two weeks and needed time to respond to
reestablish synthesis of testosterone. This hypothesis is
supported by the data of Riegle and Miller (unpublished)
that in aged male rats serum testosterone responds less
dynamically to an injection of HCG than serum testosterone
of young male rats. Aged male rats primed for several days
with HCG could release similar amounts of testosterone as
unprimed young male rats, suggesting constant or tonic
gonadotropin secretion is necessary for testicular response
to bolus injection of LHRH or HCG.

Chronic restraint stress similarly shut down
reproductive function in female rats as they ceased to
cycle and showed a persistent diestrus vaginal cytology.
Vaginal cytology is considered an indication of the stage
of the estrous cycle and reflects relative levels of
estrogen and progesterone as well as help detect ovulation
in the rat (Swartz, 1968). Since LH release was suppressed

by chronic stress, apparently little or no follicular

 

 

97

growth took place as no estrogen-induced cornified cells
were seen. The only exceptions noted were following the
second LHRH challenge when LH was present in the blood in
high concentrations. At this time vaginal cytology of 30%
of the rats was either nucleated or cornified, proestrus
or estrus respectively, which indicates LH stimulation.

It was expected that the rats in the stress groups .1
would lose weight. Whether stress itself was directly a
factor or through stress effects of glucocorticoid and
epinephrine serum concentrations effect on appetite and
metabolism was not explored. Stress animals were also
removed from food and water for a total of four hours a
day, while control rats had continued access to food and
water. Although rats normally do not eat during the day,
they do have sleep and activity periods that are inter-
rupted. It also took 45 minutes to an hour following a
stress period for the stressed rats to groom and settle
down, which could further upset their rest-activity

schedule.

 

 

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