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This is to certify that the

thesis entitled

STRESS EFFECTS ON PREGNANCY,
FETAL NORMALCY AND PROLACTIN RELEASE
IN THE LABORATORY RAT

presented by

Donald Wallace McKay

has been accepted towards fulfillment
of the requirements for

Ph.D. Animal Science

Jegree in

WA HA

V
Major professor fl

 

 

Date April 15, 1981

 

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STRESS EFFECTS ON PREGNANCY,
FETAL NORMALCY AND PROLACTIN RELEASE

IN THE LABORATORY RAT

By

Donald Wallace McKay

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Animal Science

1981

ABSTRACT
STRESS EFFECTS ON PREGNANCY,

FETAL NORMALCY AND PROLACTIN RELEASE
IN THE LABORATORY RAT

by

Donald Wallace McKay

The effect of restraint stress on serum prolactin and reproduction
was studied in pregnant Long—Evans rats. The female progeny of
stressed and non-stressed dams were evaluated for developmental normal-
cy of growth, ovarian cyclicity, fertility and fecundity. In addition,
temporal changes in serum prolactin concentrations and dopaminergic
neuronal activity in selected brain regions were determined over a 24
hr period in pregnant and pseudopregnant rats.

In the first study, restraint stress treatments were administered
to pregnant females at intervals during the 22 day gestation period.
Pregnant rats were restrained from 0100-0400 hrs on days 4 through 7
of gestation. In contrast to non-stressed controls, no nocturnal
surge of prolactin was detected in blood samples drawn at 0400 hrs on
days 4 or 7 of pregnancy. The stress treatment although severe enough
to interfere with the prolactin surge had no effect on gestation
length, abortion rates, or live litter size. Offspring from these
pregnancies experienced no deficiencies in weight or survivability
when measured at 0, l4 and 28 days of age. In the next trial, re-

straint was administered from 1300 through 1600 hrs on days 4—7, 8-11

Donald Wallace McKay
or 12-15 of pregnancy. Serum prolactin concentrations measured in
samples collected at 0300 hrs following the first and fourth days of
the respective treatment interval were not affected by restraint
stress. No effect of stress was detected on the length of gestation,
incidence of abortion or live litter size in any of the groups tested.
Prenatal stress did not influence the age of pubertal vaginal opening
in randomly selected offspring from the treatment groups. The ferti-
lity of prenatally stressed females was not different than prenatally
non-stressed controls. In the third stress trial, restraint was
applied twice daily from 0900-1100 hrs and 1300-1500 hrs on days 10-
13, 14-17 or 18-21 of pregnancy. The treatment administered on days
10-13 of pregnancy resulted in a significant incidence of abortion,
while the length of gestation was significantly increased in females
restrained on days 14-17 of pregnancy. The pregnancies of animals
treated on days 18-21 were unaffected by restraint. Upon maturity,
offspring from these pregnancies were all capable of producing normal
litters. These results demonstrate that although certain stress
regimes can terminate pregnancy, the stress induced alteration of the
nocturnal PRL surge is not sufficient to cause abortion. Furthermore,
prenatally stressed female offspring are as developmentally normal as
non-prenatally stressed controls in terms of growth, fertility and
fecundity.

In the second study, temporal changes in prolactin and LH concen-
tration and dopamine neuronal activity in selected brain regions were
determined over a 24 hour period in pregnant, pseudopregnant and
diestrous rats. Two daily surges of prolactin were observed on day 6

of pregnancy and pseudopregnancy, but not in diestrous rats.

Donald Wallace McKay
Similarly, biphasic changes in the rate of DOPA accumulation were
observed in the median eminence, but not in other brain regions, of
pregnant and pseudopregnant rats, while there were no changes in
diestrous rats. These results suggested a specific activation of
tuberoinfundibular dopamine (TIDA) neurons during pregnancy and
pseudopregnancy. Manipulation of the surge release of prolactin by
restraint stress or ovariectomy following cervical stimulation re-
sulted in changes in the rates of DOPA accumulation specific to the
‘median eminence. Restraint stress during the expected nocturnal
prolactin surge of pregnancy from 2300 hrs day 5 until 0300 hrs day 6
delayed the appearance of the surge between 3 and 6 hours. The peak
rate of DOPA accumulation in the median eminence was likewise delayed
by restraint stress. Ovariectomy following cervical stimulation
eliminated both the diurnal prolactin surge and the subsequent in-
crease in median eminence DOPA accumulation. These results suggest
that specific alterations of TIDA neuronal activity during pregnancy
and pseudopregnancy may be related to the surge release of prolactin

from the anterior pituitary.

DEDICATION

I dedicate this thesis to my wife, Kathleen-

ii

ACKNOWLEDGEMENTS

I extend my gratitude to my advisor, Dr. G.D. Riegle, for his
support, encouragement, guidance and kindness throughout this project.
Additionally, I am indebted to Dr. K.T. Demarest for his contribu—
tions to the planning and execution of a major portion of this study.
Dr. K.E. Moore provided much expertise in the interpretation of the
data, and was generous with the use of his laboratory. These experi-
ments were facilitated by the expert technical assistance provided by
Dorothy L. Okazaki, Mirdza Gramatins, Jennifer L. Miller and Susan
Stahl. Diane Hummel is gratefully acknowledged for her expert pre-
paration of this manuscript. Equipment and facilities for the produc—
tion of graphics in this thesis were kindly provided by J.M. Lipsey.

I would also like to thank the remaining members of my graduate
committee for their openness and efforts in my behalf: Drs. J.R.
Brunner, M.G. Hogberg and E.R. Miller.

The author is grateful for the financial support provided by the
Department of Animal Husbandry and through the National Science Found-
ation.

The cooperation of the entire staff at the Endocrine Research Unit
was appreciated, and special thanks are due Mrs. Alan Cleeves and Dr.

W.R. Dukelow for their assistance with logistical matters.

iii

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Page
LIST OF TABLES vii
LIST OF FIGURES viii
INTRODUCTION 1
CHAPTER 1 4
Reproduction in the Laboratory Rat 4
Hormones of Pregnancy 5
Maintenance of the Corpus Luteum 6
Hormone Regulation of Prolactin Surges 6
Uterine and Placental Influences on Pregnancy and

Pseudopregnancy 7
CHAPTER 2 9
Stress Effects on Reproduction and Developmental Normalcy--- 9
Endocrine Response to Stress 9
Stress Effects on Prolactin and LH Release 9
Stress Effects on Reproduction 10
Prenatal Stress and Developmental Normalcy 12
Materials and Methods 15
Animals, Housing and Routine Handling 15
Breeding 16
Blood Collection 16
Restraint Treatment 16
Radioimmunoassay 17
Statistical Analyses 17
Results 17

The Effect of Restraint Stress on the Nocturnal Surge
of Prolactin and Reproduction in Long-Evans Rats ----- 17

The Effect of a 3 hr Restraint Stress Administered on
Days 4-7, 8—11, or 12-15 of Pregnancy 18

iv

TABLE OF CONTENTS (continued)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Page
The Effect of Intermittent Restraint Stress on Repro-

duction During Late Pregnancy in the Rat 24
Discussion 33
CHAPTER 3 38

Prolactin Release and Tuberoinfundibular Dopaminergic Nerve
Activity During Pregnancy and Pseudopregnancy 38

Prolactin, Dopamine and Tuberoinfundibular Dopaminergic
Neurons 39
Dopamine in Portal Vasculature 4O
Hormonal Effects on TIDA Neuronal Activity 41
Stress and Biogenic Amines 44
Materials and Methods 51
Animals - Housing and Handling 51
Tissue Handling and Assay—DOPA Accumulation 52
DA Steady-State Concentration 53
Blood Collection 53
Statistical Analyses 53
Results 54

 

Characteristics of Tuberoinfundibular Dopaminergic
Neuronal Activity During the Daily Surges of Prolac—
tin Secretion in Pregnant and Pseudopregnant Rats---- 54

Effects of Blood Sampling on Prolactin During Pregnancy 70

The Effect of an Acute 4 hr Restraint Stress Admini-
stered During the Nocturnal or Diurnal Surge of Pro-
lactin and DOPA Accumulation in Pregnant and Pseudo-
pregnant Rats 72

 

The Effect of Restraint Stress During the Nocturnal
Prolactin Surge of Pregnancy on the Pattern of Pro-
lactin Release 75

 

The Effects of Restraint Stress During the Nocturnal
Prolactin Surge of Pregnancy on the Rate of DOPA
Accumulation 87

 

The Effect of Restraint Stress During the Nocturnal
Prolactin Surge of Pregnancy of the Rate of DOPA Ac—
cumulation at Selected Times After Stress 90

 

The Effect of Ovariectomy on Prolactin Secretion and
Anterior Pituitary Dopamine Content in Cervically
Stimulated Rats 93

 

TABLE OF CONTENTS (continued)

 

 

 

 

 

Page

The Effect of Ovariectomy Following Cervical Stimula-
tion on DOPA Accumulation in the Median Eminence ------- 96
Discussion 99
CHAPTER 4 107
Summary 107
Stress Effects on Pregnancy and Offspring Development-- 109
Prolactin Release and TIDA Neuronal Activity 109
BIBLIOGRAPHY 112

vi

TABLE

10

LIST OF TABLES

 

 

 

Page
Effect of Restraint Stress Administered from 0100-0400
hrs on Days 4-7 of Pregnancy 21
Effect of Restraint Stress Administered from 1300-1600
hrs on Days 4-7, 8-11, or 12—15 of Pregnancy 27
Days to Vaginal Opening of Prenatally Stressed Female
Rats 28
Fertility of Prenatally Stressed Female Rats 29

 

Effect of Restraint Stress Administered Twice Daily
from 0900-1100 hrs and 1300-1500 hrs on Days 10-13, 14-
17, or 18-21 of Pregnancy 31

 

Fertility and Fecundity of Prenatally Stressed Female
Rats 32

 

DOPA Accumulation in Selected Brain Regions on Day 7 of
Pregnancy 68

 

DOPA Accumulation in Selected Brain Regions on Day 7 of
Pseudopregnancy 69

 

DOPA Accumulation at 1200 hr in Pregnant, Pseudopreg-
nant and Diestrous Female Rats 71

 

Number of Rats Per Treatment Cell for Determination of
DOPA Accumulation Following Cervical Stimulation-
Ovariectomy 100

 

vii

FIGURE

10

ll

12

13

LIST OF FIGURES

The effect of 4 days of 3 hr of restraint stress
treatments initiated on day 4 of pregnancy on serum
prolactin concentrations

 

The effect of prenatal stress on the growth of off-
spring

 

The effect of 4 days of 3 hr of restraint stress treat-
ments initiated on days 4, 8 or 12 of pregnancy on
serum prolactin concentrations

 

Distribution of major DA neuronal systems in the rat
brain

 

Schematic diagram of a tuberoinfundibular dopaminergic
neuron

 

The pattern of prolactin and LH in the pregnant rat----

The pattern of prolactin and LH in the pseudopregnant
rat

 

The pattern of prolactin and LH on the second day of
diestrous

 

The pattern of DOPA accumulation in the median eminence
in the pregnant rat

 

The pattern of DOPA accumulation in the median eminence
in the pseudopregnant rat

 

The pattern of DOPA accumulation in the median eminence
on the second day of diestrous

 

Effect of orbital sinus puncture on serum prolactin
concentrations during early pregnancy

 

The effect of restraint stress on the nocturnal surge
of prolactin and DOPA accumulation in the pregnant rat—

viii

Page

19

22

25

42

45

56

58

6O

62

64

66

73

76

LIST OF FIGURES (continued)

FIGURE

14

15

l6

17

18

19

20

21

22

 

Page
The effect of restraint stress on the nocturnal surge
of prolactin and DOPA accumulation in the pseudopreg-
nant rat 78

The effect of restraint stress on the diurnal surge of
prolactin and DOPA accumulation in the pregnant rat--—- 80

The effect of restraint stress on the diurnal surge of
prolactin and DOPA accumulation in the pseudopregnant
rat 82

 

The effect of a 4 hr restraint stress on the pattern of
prolactin release in the pregnant rat 85

 

The effect of a 4 hr restraint stress of the pattern of
DOPA accumulation in the median eminence of the preg-
nant rat 88

 

Effect of a 4 hr restraint stress on DOPA accumulation
at selected times 91

 

The effect of ovariectomy following cervical stimula-
tion on the pattern of prolactin release 94

 

Anterior pituitary dopamine concentrations in ovariec—
tomized-cervically stimulated rat 97

 

The effect of ovariectomy following cervical stimula-
tion on the pattern of DOPA accumulation in the median
eminence 101

 

INTRODUCTION

Stress is an oft used, but poorly defined concept. While commonly
stress is associated with anxiety or trauma, it may be more accurately
described as an organism's attempt to maintain homeostasis when con-
fronted by novel or noxious stimuli. Since it is recognized that both
psychological and physical stressors can affect many body functions,
many management decisions in the livestock industry are made in an
attempt to reduce stress. Yet without an understanding of the mecha-
nisms by which stress produces alterations in the body's processes,
any management decisions will be lacking a sound basis.

The stress response is mediated by a number of neuroendocrine
reactions. The most thoroughly documented endocrine response to a
stressor is the sustained release of adrenal corticoids. While the
increased release of adrenal corticoids is a reliable index of stress,
it is now apparent that stress can influence the endocrine control of
most body functions.

A number of studies has confirmed that stress can interfere with
reproduction, however, the mechanisms through which stress acts to
inhibit reproductive processes are unknown. The identification of
stress induced alterations in the hormonal regulation of reproduction
will provide knowledge of the physiology of reproduction, and give as
a basis from which to make decisions regarding the management of

stress.

2

To study the effects of stress in the pregnant female, one must
not only consider the deleterious consequences of stress on the health
of the dam or the success of the pregnancy, but it is important to
assess the normalcy of the offspring that were stressed in 35239,
Previous reports have indicated that such offspring suffer from
various "emotionality" disturbances and altered sexual behavior, but
studies on functional reproductive capabilities of these animals are
lacking.

Throughout this thesis, major emphasis will be given to the
effects of restraint or stress on reproduction in the rat. The rat
offers several distinct advantages over other species in the study of
reproduction: 1) Fertility - the laboratory rat under standard condi-
tions is capable of producing litters of consistent size in a short
time; 2) management - because of their size and short estrous cycle
length, the routine husbandry and breeding of laboratory rats is
simplified; and 3) background knowledge - the major reason for the use
of the laboratory rat in the study of reproduction is the abundance of
information available on the hormonal support of pregnancy in this
species.

The present investigation had the following specific aims:

1) To examine the effects of stress on the maintenance of preg-
nancy, with regard to the hormonal environment sustaining
the pregnancy.

2) To determine the normalcy of the development and function of

offspring that were stressed prenatally.

3
3) To more thoroughly characterize stress effects on prolactin
release during pregnancy, and to associate those changes
with specific alterations in neuronal activity within the
hypothalamus.

Chapter 1 will be an overview of reproduction in the rat with
particular emphasis on the hormonal control of pregnancy and pseudo-
pregnancy. Included in this chapter is a brief review of physiology
to acquaint the reader with terminology and current thought.

Stress effects on pregnancy and normalcy of offspring will be the
subject of Chapter 2. In addition to a review of the literature in—
volving stress effects on hormone release, pregnancy, and offspring
development, this section will include original work that addresses
the first two objectives of the thesis.

The next chapter focuses on the regulation and release of prolac-
tin during pregnancy and pseudopregnancy. The literature surveyed in
Chapter 3 selectively deals with a possible catecholaminergic control
of prolactin release, and the hypothesis that dopamine is a major
inhibitor of pituitary prolactin secretion. The results of this
section provide additional insight and explanation for the findings in
the previous chapter.

Finally, Chapter 4 is a brief summary and discussion of the

aforementioned studies.

CHAPTER 1

Reproduction in the Laboratory Rat

The reproductive "normalcy" of a laboratory rat is often esta-
blished by observing the pattern of estrous cycles. In the labora-
tory, this is accomplished by the use of a vaginal smear or wet prep
followed by microscopic examination of the obtained cells. Changes in
vaginal histology very accurately reflect alterations in ovarian
hormone secretion such that diestrus is characterized by the presence
of leucocytes in the smear, while proestrus and estrus preps typically
show an abundance of nucleated and cornified epithelial cells, respec—
tively (Nalbandov, 1964). Lack of a functional corpus luteum (C.L.)
accounts for the short cycle length of 4-5 days in the laboratory rat
(Bone, 1979) compared to the 21 day estrous cycle of the pig or cow
which form functional corpora lutea in each ovarian cycle. Stimula-
tion of the uterine cervix during proestrus, estrus, or the first day
of diestrus triggers neuroendocrine mechanisms which may result in the
development of functional corpora lutea (Long and Evans, 1921). If
stimulation of the cervix occurs during copulation with a fertile
male, then corpora lutea of pregnancy are formed and maintained for
approximately 22 days, the normal gestation period in the rat.

If cervical stimulation is via artifical means (e.g., glass rod,

or electrical stimulation) or by contact with an infertile male, then

5
corpora lutea of pseudopregnancy are formed which usually regress 12
to 14 days later. Thus, CL regression or luteolysis can be temporari-
ly delayed for 2 to 3 weeks by artifical cervical stimulation or

natural mating, respectively.

Hormones of Pregnancy

 

The most pronounced alteration in hormone secretion occurring
during pregnancy and pseudopregnancy is increased progesterone release
from the corpus luteum. Several years ago, it was reported that the
function of the corpus luteum was dependent on pituitary luteotropic
stimulation (Astwood, 1941). In recent years it has been shown that
the primary pituitary luteotroph is prolactin and the luteotrophic
function of prolactin is dependent on the stimulatory function of two
daily surges of hormone secretion (Butcher £3 31,, 1972; Freeman gt
a},, 1974; Smith 35 al,, 1975). The surges of prolactin occur twice a
day in pregnant and pseudopregnant rats such that one surge (noctur-
nal) appears in the early morning prior to the illumination of the
Vivarium, and a smaller peak (diurnal) is detected just prior to
lights off (Butcher 3E_§l,, 1972; Freeman 95 al., 1974).

Early studies of this endocrine control system demonstrated that
increased pituitary prolactin release on the second day of diestrus
could maintain functional corpora lutea (Nikitovitch-Winer and
Everett, 1958) and that prolactin (PRL) supplementation could maintain
pregnancy in hypophysectomized rats (Astwood, 1941). Pharmacological
blockade of the prolactin surges on day 2 of pseudopregnancy resulted
in the precipitous decline in serum progesterone concentrations and
the regression of the corpora lutea (Smith gt_al,, 1976). These

results suggest that the surge release of prolactin on day 2 of

6

pregnancy or pseudopregnancy is responsible for the "rescue" of the

C.L. from regression or luteolysis (Smith_g£.§l., 1975).

Maintenance of the Corpus Luteum

 

In addition to prolactin, other hormones are involved in the C.L.
maintenance of pregnant and pseudopregnant rats. A number of studies
have demonstrated a role for LH in sustaining the corpora lutea (Roth-
child gt_§l,, 1974; Morishige and Rothchild, 1974a; Lam and Rothchild,
1977). Current evidence suggests that the function of LH is to stimu-
late the luteal production of androgen, which is subsequently aroma-
tized to estrogen within the C.L. (Gibori 35 31., 1978). The result-
ant estrogen synergizes with prolactin to sustain luteal progesterone
(Gibori gt_§l,, 1977; Gibori and Keyes, 1978; Gibori and Richards,

1978; Gibori e_t_il_., 1979).

Hormone Regulation of Prolactin Surges

 

The importance of prolactin for the initiation and maintenance of
luteal function necessary to sustain pregnancy in the rat cannot be
overstated. The interrelationships between ovarian steroids and
prolactin release were recognized early. The induction of pseudopreg-
nancy is most readily accomplished during stages of the estrous cycle
when estrogen and progesterone are elevated (Greep and Hisaw, 1938).
Additionally, pseudopregnancy could be achieved by single injections
of estrogen or progesterone, or multiple prolactin injections in the
absence of cervical stimulation (Alloiteau, 1957; Alloiteau and

Vignal, l958a,b).

7

Maintenance of the surge release of prolactin is affected by
ovarian steroid secretion. Ovariectomy (OVX) following cervical
stimulation causes the abolition of the diurnal prolactin surge and an
attenuation of the nocturnal surge and its premature disappearance
after day 7 (Freeman g£_§l,, 1974). Replacement therapy of progester-
one restores the nocturnal surge release of prolactin in the OVX rat,
but the diurnal surge remains absent (Freeman and Sterman, 1978).
Estradiol implants in the OVX cervically stimulated female reinstate
the diurnal surge but lessen the magnitude of the nocturnal surge.
These findings demonstrate the importance of the ovary in the normal
secretion of prolactin of pseudopregnancy. Steroid hormones, however,
are not required for the initiation of prolactin surges induced by
cervical stimulation, as surge concentrations of serum prolactin have
been achieved in ovariectomized-adrenalectomized rats (Smith and

Neill, 1976a).

Uterine and Placental Influences on Pregnancy and Pseudopregnancy

Although the successful maintenance of pregnancy depends upon the
secretion of progesterone from the corpus luteum (Csapo and Wiest,
1969), pituitary release of prolactin is only necessary to maintain
pregnancy the first eleven days of gestation (Pencharz and Long,
1931).

Rat placental luteotrophin (rPL) a prolactin-like compound from
the placenta is first measurable on day 8 of pregnancy. During the
latter half of the 22 day pregnancy in the rat, rPL is the predominant
luteotrophin (Gibori and Richards, 1978). The diurnal and nocturnal

pituitary surges disappear coincident with the development of placental

8
luteotrophin secretion. It is speculated that rPL may feedback to the
hypothalamus to cause the early suppression of prolactin surges during
pregnancy (Yogev and Terkel, 1978). During pregnancy, the nocturnal
surge is apparent for 10 days, while the diurnal surge endures for
only the first 8 days (Smith and Neill, 1976b). However, in pseudo-
pregnant rats, the nocturnal surge persists through day 11 while the
diurnal surge disappears a day earlier.

Earlier findings demonstrated that hysterectomy (HYST) prevented
the regression of the C.L. (Hashimoto 35 31., 1968; Hausler and Mal-
ven, 1971), which suggested that uterine or placental factors may
regulate the disappearance of the prolactin surges. Removal of the
uterus but not the ovaries of cervically stimulated rats results in
the maintenance beyond 16 days of both nocturnal and diurnal surges
(Freeman, 1979). Although the uterus produces a substantial amount of
prostaglandin an, a potent luteolytic agent that has its action
directly on the C.L., there appears to be a uterine factor that acts
within the CNS to suppress the surge release of prolactin. OVX-HYST
rats supplanted with progesterone experienced both surges at day 16,
while the diurnal surge was absent in progesterone treated OVX rats
with intact uteri. In pregnant rats, progesterone is capable of
reinitiating the nocturnal PRL surge up to 4 days after its normal
cessation provided the uterine-placental unit is removed (Voogt,
1980). Thus, it appears that the uterus produces a substance that
works independently of any luteolytic action on the C.L., and that
this factor acts to terminate the nocturnal release of PRL via an

undetermined neuronal mechanism.

CHAPTER 2
Stress Effects on Reproduction and Developmental Normalcy

Endocrine Response to Stress

 

Numerous observations have shown that the scope of the endocrine
response to stress is not limited to the hypothalamo-hypophysial-
adrenal axis (Ajika g£“§13, 1972; Brown-Grant, 1954; Krulich gt al.,
1974). It was suggested that the adrenals may act to modify the
release of the pituitary hormones (e.g., LH, PRL) as pharmacological
doses of glucocorticoids (50 ug dexamethasone/100 g body wt) have been
shown to lessen the stress induced release of some pituitary hormones
(Euker gt 91., 1975; Harms g£_§l,, 1975). However, the adrenal glands
are not necessary for the manifestation of many other stress responses
such as decreased TSH secretion or inhibition of ovulation, and they
may have little physiological function in the mediation of acute
stress effects on pituitary hormones other than ACTH (Brown-Grant,
1954; McKay 25 31., 1975).

A number of studies have demonstrated that the magnitude and
direction of the hormonal changes due to stress are dependent upon
such factors as age, reproductive status, circadian cyclicity, and the
intensity and duration of the stressor (Dunn 3; al., 1972; Morishige

and Rothchild, 1974b; Riegle and Meites, 1976a,b; Tache 35 31., 1976).

Stress Effects on Prolactin and LH Release

 

Since prolactin release was demonstrated to be "stress susceptible"

(Grosvenor gt al., 1965), a number of studies have been conducted to

10
determine the effects of acute stress on serum prolactin concentra-
tions. Such routine laboratory procedures as blood sampling were
shown to result in significant elevations of prolactin concentrations
in female rats (Neill, 1970). Acute ether exposure and blood sampling
evoked dramatic increases in serum PRL and LH in intact male rats,
while the same stress in gonadectomized males resulted in a decrease
in LH levels and a concomitant elevation of serum PRL concentrations
(Euker g£_§l,, 1975).

The reported increases of adenohypophysial hormones other than
ACTH, which followed "stress" were in apparent contradiction to Selye's
"hypophyseal shift theory" (Selye, 1946). Based on morphological and
functional criteria, it was predicted that the "general adaptation
syndrome" or stress response would result in depressed release of all
pituitary hormones other than ACTH, as such functions as growth,
sexual development, and thyroid activity were shown to be decreased
when animals were subjected to a chronic stress treatment.

Indeed, it was subsequently demonstrated, that repeated exposures
to the stressor, over a period of days resulted in decreased release
of LH, PRL, growth hormone (GH), and follicle stimulating hormone
(FSH), while corticosterone concentrations were elevated (Tache gt
.31., 1976; Riegle and Meites, 1976b). These results demonstrate
profound differences in the pituitary response to short or acute

stressors versus a repeatedly or chronically stressful environment.

Stress Effects on Reproduction

 

A number of reproductive processes in the female are believed to

be affected by stress. Stress has been shown to decrease or block

11
ovulation (Terman, 1973; Hagino g£_§l,, 1969; McKay gt 31., 1975),
while fertilizability of oocytes appears to be relatively stress
resistant (Ulberg and Sheean, 1973; Sod—Moriah, 1971). Studies con-
sidering its effects of stress on egg and embryo development after
fertilization indicate variable alterations in reproductive function.
Euker and Riegle (1973) studied the effects of restraint stress admini—
stered over a number of days on pregnancy. Reproduction was affected
by stress in an "all or none" fashion; the females aborted, or they
gave birth to a litter of normal size.

It was demonstrated that increased adrenal activation was detri-
mental to pregnancy, as increased body temperature caused embryonic
degeneration in intact females, but not adrenalectomized rats (Fernando-
Cano, 1958) or ewes (Tilton gt 31., 1972). Decreased numbers of
implantation sites were evident when ACTH or corticoids were admini-
stered prior to nidation in rats (Yang g£_al,, 1969). The maintenance
of ACTH at high levels regardless of blood glucocorticoid concen-
trations resulted in decreased pup and litter size in mice (Kittinger
g£_§l,, 1980). This suggests that ACTH may affect reproductive func-
tion independent of its effect on the adrenals. In contrast to the
study of Kittenger 32 a1, (1980), we found that adrenalectomized
pregnant rats maintained a normal pregnancy (McKay and Riegle, 1978).
These results indicate that increased ACTH, due to adrenalectomy, was
not sufficient to disrupt pregnancy.

Restraint stress administered to adrenalectomized rats on days 1-
4 of pregnancy resulted in a total loss of pregnancy associated with a

precipitous decline in serum progesterone concentrations (McKay and

12
Riegle, 1978). Similar stress treatment of intact pregnant females
had no effect on pregnancy, and resulted in increased serum progester-
one levels. These findings suggest that adrenal progesterone may have
an important function in the maintenance of pregnancy.

The aforementioned findings demonstrated drastic alterations of
pituitary and adrenal hormones due to stress. Whether or not these
hormonal alterations act centrally to affect pregnancy or have a peri—
pheral mode of action has not been established. Experimental altera-
tion of the uterine vasculature has resulted in embryonic malforma-
tions (Brent and Franklin, 1960), and increased embryonic mortality
(George g£_gl,, 1967; Senger gt 31., 1967). However, it appears that
the uterus is relatively resistant to sudden changes in circulation
during pregnancy (Bruce, 1972). The resistance may be explained in
part by the development of collateral circulation (Bruce, 1967), or by
circulatory changes within the fetus to aid its continued survival

(Assali and Brinkman, 1973).

Prenatal Stress and Developmental Normalcy

 

In addition to stress induced embryonic mortality that may arise
during pregnancy, it is important to consider prenatal induced stress
anomalies manifest in fetal or postnatal development (Euker and
Riegle, 1973). Since Thompson (1957) first demonstrated the effects
of prenatal psychological stress on offspring emotionality, many
investigators have attempted to study the phenomenon. Archer and
Blackmun (1971) reviewed the literature on this subject, and summa-
rized that due to differences in: 1) sex, strain and species; 2) the

prenatal manipulation; and 3) the completeness of the description of

13
behavioral changes, very few conclusions could be drawn except that
"some change in activity—reactivity may be induced".

Behavioral abnormalities coincident to prenatal immobilization
stress have been reported. Adult prenatally stressed males exhibit
less mounting behavior (i.e., demasculinized) (Ward, 1972; Herrenkohl
and Whitney, 1976), and display a higher incidence of lordosis in
response to other males (i.e., femininized) (Ward, 1972; Dahlof gt
31,, 1977). Some of the observed changes of offspring development,
however, may be due to stress effects on the mothers' nursing ability
(Herrenkohl and Whitney, 1976).

Prenatal physical stress or glucocorticoid treatment have been
implicated in such physical changes as delayed postnatal growth (Barlow
‘gE_§l,, 1978; Reinisch 35 al., 1978), cleft palate (Barlow g£_§l,,
1975) and decreased ano-genital distances of male rat pups (Dahlof 35
31,, 1978a,b). Neuroendocrine reflexes have been reported altered by
prenatal stress, as ether stress administered to adult prenatally
stressed males resulted in only a small increase in prolactin secre-
tion in contrast to control animals (Politch 35 al., 1978), some
investigators believe that developmental behavioral changes such as
feminization and demasculinization may be due to an altered androgeni-
zation of the fetal rat brain (ward and Weisz, 1980), as maternal
restraint stress during late gestation alters the occurrence of peak
levels of testosterone within the male fetus (Ward and Weisz, 1980;
Weisz and Ward, 1980).

Chapman and Stern (1978, 1979) issued a caveat to the interpreta—

tion of maternal stress studies: Failure to control for the litter

14
variable, e.g., litter size, male/female pup ratio, genetic lineage,
may account for previously reported effects of prenatal stress. In
studies that followed similar stress regimen as that of Ward (1972)
and Herrenkohl and Whitney (1976), it was found that litter variance
in the analysis accounted for most of the observed changes. Under
conditions of controlled litter size, balanced for sex, the removal of
litter variance eliminated any statistical significance that otherwise
would have been attributed to the stress treatment. These investiga-
tors further cautioned against the use of low animal numbers and
suggested that the use of littermates in the same treatment group be
avoided.

While less data exist for prenatal stress effects on the subse—
quent development of females, one laboratory has demonstrated de-
creased fertility and fecundity in such animals (Herrenkohl, 1979;
Herrenkohl and Gala, 1979). Adult prenatally stressed females dis—
played a significant inability to nurse litters past day 10 of lacta-
tion, even though the litter size in these animals was smaller than in
non-stressed control females. Behaviorally, prenatally stressed
female mice appeared to be less aggressive toward intruder males, and
it was speculated that this is to compensate for a decreased attracti-
vity to males (Politch and Herrenkohl, 1979).

Taken together, the findings presented in this literature review
do not provide a clear understanding of stress effects on hormone
release or reproductive control mechanisms. These discrepancies that
seem to characterize the entire study of stress relate to major
problems: 1) the use of a poorly defined stressor and lack of a

complete description of stress effects and 2) the individual and

15
strain differences in the responsiveness to stressors. To overcome
these problems, it is necessary that investigators utilize more care-
fully controlled experiments in which a measurable and repeatable
stressor is administered to the animal. Objective measures of the
stress response must be used, such that individual variation may be
more easily partitioned.

The purpose of this series of studies is to l) examine the
effects of a chronic restraint stress on maternal prolactin concentra-
tions, and on the ability of pregnant rats to sustain a normal preg-
nancy, and 2) assess the development and/or reproductive competence of

prenatally stressed female offspring.

Materials and Methods

Animals, Housing and Routine Handling

 

Long—Evans rats were housed in a temperature (23:2°C) and light
controlled vivarium (lights on from 0600 to 1800 hrs) for at least two
weeks prior to experimentation. Male and female rats were housed 3
and 4 rats per box, respectively, and were allowed free access to food
(Wayne Lablox) and water except during restraint treatments. Females
for any given experiment were of similar age and background. Daily
vaginal lavage and cytological evaluation were used to determine each
rat's reproductive status with regard to pregnancy, breeding, or
ovarian cyclicity. All female rats were handled daily for this pur-
pose a minimum of two estrous cycles prior to experimentation, and

continued until day 16 of pregnancy.

16
Breeding
On the afternoon of proestrous as determined by the vaginal wet
prep, one adult female was placed in a box of males housed 3 per cage.
The next morning upon return to her home cage, a vaginal wet prep was
obtained and observed microscopically for the presence of sperm. The

detection of sperm established Day 1 of pregnancy.

Blood Collection

 

Blood samples were collected via orbital sinus puncture under
light ether anesthesia at various times as specified in Results. Control
samples were collected within 90 seconds of cage disturbance to minimize
the effects of bleeding on serum hormone concentrations.

The resultant blood samples were allowed to clot and were refriger-
ated at 4°C overnight. The clotted samples were centrifuged at 4°C
for ten minutes to separate serum from cellular elements. Individual
samples were stored in disposable glass culture tubes at -20°C for
several days until radiommunoassays were performed for the measure-

ments of prolactin.

Restraint Treatment

 

Restraint stress was achieved by securing ether anesthetized rats
with tape to a stainless steel counter top. The rats were thus main-
tained in a supine position for intervals as specified in Results.
During the stress interval, the animals were physically disturbed
every 10-20 minutes to prevent acclimation to the restraint. Non-
stressed controls remained undisturbed in their cages during the same

time interval. Any restraint or blood sampling conducted between

17
1800-0600 hrs was facilitated by the illumination of two red 25 watt

incandescent bulbs.

Radioimmunoassay

 

Double antibody RIA determinations of PRL and LH serum concentra—
tions were made with the aid of kits supplied by Dr. A.I. Parlow of
the NIAMDD, and a second antibody, ovine anti-rabbit gamma globulin,
developed at the Endocrine Research Unit. Hormone values measured in
sera, are expressed in terms of NIAMDD rat prolactin RP-l and NIAMDD
rat LH RP—l. All blood samples were run in duplicates of two dilu-
tions and samples from any particular experiment were processed in the

same assay to limit variability.

Statistical Analyses

 

Frequency of abortion was determined by Chi—square adjusted for
small n (Sokal and Rohlf, 1969; Rohlf and Sokal, 1969). Differences
in the length of gestation, litter size comparisons, number of days
until vaginal opening and comparisons of serum prolactin concentra-
tions were evaluated by use of Student's_petest (Sokal and Rohlf,
1969; Rohlf and Sokal, 1969). Weight of litters was evaluated by a
two-way analysis of variance by use of the Program Balanova supplied

by the MSU computer laboratory.

Results

The Effect of Restraint Stress on the Nocturnal Surgg of Prolactin
and Reproduction in LongrEvans Rats

 

 

In the first study, the restraint was administered between 0100

and 0400 hrs on days 4-7 of pregnancy. (Preliminary data in our

l8
laboratory established the existence of an early morning (nocturnal)
surge of prolactin at this time.) Serum prolactin concentrations were
decreased (p<.001) from control when measured at the end of the re—
straint period on days 4 and 7 of pregnancy (Figure l). The four day
stress treatment did not block the occurrence nor the magnitude of the
diurnal prolactin surge as measured at 0100 hrs on day 7 of pregnancy.
Although the acute effect of stress on serum prolactin was pronounced,
this particular stress regimen had no effect on abortion, litter size
or gestation length (Table 1). Within 16 hours of parturition, the
litters were weighed and sexed. The litters were reduced to 3 males
and 3 females. No significant differences of weight were detected
between control and prenatally stressed litters when measured at birth
14 or 28 days of age (Figure 2).

The Effect of a 3 hr Restraint Stress Administered on Days 4-7, 8-11,
or 12—15 of Pregnancy

 

 

It is established that restraint stress administered in the
afternoon during early pregnancy or pseudopregnancy is capable of
decreasing the magnitude of the diurnal prolactin surge (Riegle and
Meites, 1976b; Freeman 35 al., 1974). Although previous work sug-
gested that the nocturnal surge of prolactin was not affected by
stress (Freeman g£_al,, 1974), our work demonstrated that restraint
stress administered during the time of the expected surge could inter-
fere with its occurrence. To further explore the effects of restraint
stress on reproduction and the nocturnal surge of prolactin, the
following experiment was conducted.

Pregnant female rats were subjected to restraint stress from

1300-1600 hrs on days 4-7, 8-11, or 12-15 of pregnancy. A blood

19

.mm

H muammmuaou amp some doom mafia Hmuauuo> one .Amancv mamaecm vmmmouum uafimuum
low we mam>ma cHuomHoum some men meadow mums vowmnm mnu mam .Aqauav maouuaoo
mmouumiaoa mo macaumuuaoocoo sfiuomaoua amoa uaomouaou when ammo 65H .ucoaumouu
mo mane nunsow mam umufiw one do mmmuum umumm can ouommn showcase macaw Houwnuo
he wouooaaoo moHQEMm wooan CH woawaumumv 6903 meowumuucoocoo afiuomaoum Epsom

.mnoaumuucoocoo afiuomaoum Eamon co mocmawmum
mo q wmw co wmumfiuficfi muamaumouu mmouum uafimuummu an m mo ammo q no uoommo 05H

.H munmam

20

 

 

 

 

 

 

 

 

 

 

 

 

 

 

o
9.

O
O
N
(wmas W 5U) NIlOV'lOHd

O
0
r0

0
0
¢

 

OOm

21

TABLE 1

Effect of Restraint Stress Administered from 0100-0400 hrs
on Days 4-7 of Pregnancy

 

 

n n Gestation Litter

abort length (days) Size
Control 14 1 22.50:.04a 11.69:.73
Restrained l3 1 22.14:.08 10.29:.98

 

aMean i S.E.

bGestation length of those pregnancies terminating with live
birth.

22

.vmzmuuuom kaamofinmmuw on ou Hanan oou mm3 Mm mnu .c3onm mum
moafla o: oumnz .mm H uammmwmmu moon mnu moum mocHH HmUHuum> m£H .Amuav muouufia
wmmmmuum waamumaoum mo muanmB some mammouaou mumn vmwmnm mam Awucv muouuaa Houu

Icoo mo munwfio3 cmoa udommuamw mumn ammo .owm mo whom mm mam «H mum3 mama mam
conB cfimwm was voafimuno coca ouo3 muouufia ozu mo mucmfioz .moamamm m mam moama
m cm confine was ooxmm whoa >oamawmua mo hue want do mu: oquIooao Boom vommouum
moamamm unmawwum no meme Houuaoo wommouumlaoa Boom muouufia .nuuan mo how map so

.wawuammmo mo nu3oum onu do mmmuum Hmumcmum mo momwmo 05H

.N musmwm

23

seam?

 

 

 

 

 

 

 

 

 

 

 

 

unfinuWN _ ON 0

_

. emu
m5<2mu muqz i

(5) 'lM HBlll‘l Nvaw

24
sample was collected by orbital sinus puncture at 0300 hrs following
the first and last day of treatment, and was subsequently assayed for
prolactin. Results presented in Figure 3 show that restraint stress
had no measurable effect on serum prolactin concentrations when
‘measured at 0300 hrs following 1 or 4 consecutive days of stress
treatment. Restraint treatments administered on days 4-7, 8-11, or
12-15 did not cause any significant changes in frequency of abortion,
gestation length, or live litter size (Table 2).

Randomly selected females were weaned from each litter at 21 days
of age. No more than two females were saved from any particular
litter. Each female was checked daily for vaginal opening and the
results of that study are presented in Table 3. When compared with
respective control females, prenatal stress did not result in any
significant change in the age at which vaginal opening was detected.
Subsequent vaginal lavage revealed no noticeable differences in
ovarian cyclic characteristics between prenatally stressed females and
controls.

Table 4 illustrates the fertility of the same female rats. When
mated to control males, female rats prenatally stressed from days 4—7,
8—11, or 12-15 of gestation showed no significant inability to bear
normal litters of healthy offspring. In fact, the only animal that
did not successfully complete pregnancy was a 12-15 control female.

The Effect of Intermittent Restraint Stress on Reproduction During
Late Pregnancy in the Rat

 

 

In a final study, the restraint interval was separated into two

2 hr periods, administered four days consecutively. Rats were

25

.Mm H mucomoumou amp sumo moum oaHH HmoHuum> use

.onmaom Ammv oommouum uaHmuumou uonow mums wovmnm was .Auv mHouucoo vommmuumlaoc
ucommuaou mumn ammo .meHumaHauouov mum mo mwmuo>m ocu mucomouaou umn 50mm .ucoa
tumouu mo whom nousom cam umuHm mam waHsoHHom on come um munuocsm moch HmanHo
mH> wmuomHHoo monamm vooHn :H vocHahouow ouoB cHuomHoHa mo mcoHumuucooaoo adumm

.mcoHumuucmocoo aHuomHoua anuom so mommawmum mo NH no w .q
mxmw no woumHuHcH muaoaummuu mmouum uaHmuumou mo u: m «0 w%mv q mo uommmo one

.m mustm

26

 

 

 

 

ens]
l2

 

 

 

 

 

 

 

 

 

 

 

250 T

O O
'2 9.
(“mm uni/DU) NllOV'IOHd

200 L

50"

 

27

.oommonum uaHmnummm H mm “Honncou u o “m:0HumH>mnnn<o

.auan m>HH nuH3 wcHumaHanou mmHoammwonn omo:u mo SuwaoH aoHumummu

 

 

 

 

 

a
.m.m H amaze
mm. nmw.m Ho. an.HH mH.nno.- on.“ m.- N o w m mHINH
oo. nmm.nn mo.HneH.oH nH.nnm.~N mn.nom.- m H m w Hnsw
mH.Hnmm.HH a-.nnmo.on mm.“ m.- aNH.HmH.~N N o w m nus
mm 0 mm 0 mm u mm o Amnaev
Hm>noucH
muHmnouuHH o>HH nowaoH aoHnmumou unon< § d uaHmnumom

n

 

umoamamonm mo mHINH no .HHIw
.mlq mnmn so one oOOHIOOMH Eonm monoumHaHav< mmmnuw ucwmnumom mo nommmm

N MHm<H

28

TABLE 3

Days to Vaginal Opening of Prenatally
Stressed Female Rats

 

Age at Vaginal Opening

 

 

 

Restraint n

Interval Control Stressed Control Stressed
4—7 9 10 39.89: .818 40.3o:1.39
8—11 10 10 40.90i1.66 40.30i1.18
12-15 10 10 38.90il.14 37.40il.29

aMean i S.E.

bPregnant rats were restraint stressed between 1300—1600 hrs
on days 4-7, 8-11 or 12-15 of pregnancy

29

TABLE 4

Fertility of Prenatally Stressed Female Ratsb

 

Litter Size

 

 

Control Stressed

4-7 12.30:.60a 12.1i.43
8-11 13.2 :.79 ll.5i.62
12-15 12.33:.53 l3.4i.58

 

gMean i S.E.

bPregnant rats were restraint stressed
between 1300-1600 hrs on days 4-7,
8-11 or 12-15 of pregnancy.

30
stressed from 0900 through 1100 hrs and 1300 through 1500 hrs on
either days 10-13, 14-17, or 18-21 of pregnancy. Table 5 shows the
effect of restraint stress on abortion, gestation length and litter
size. The frequency of abortion was significantly (p<.05) increased
in rats subjected to restraint on days 10-13 of pregnancy. Gestation
length and litter size were not affected in those females that de-
livered live young.

Rats subjected to restraint on days 14-17 of pregnancy had a
significantly greater (p<.Ol) length of gestation (23.3i.28 vs.
22.19:.13 days), but live litter size remained the same.

No differences were detected in length of gestation, litter size,
or frequency of abortion between control and restraint stressed rats
handled on days 18-21 of pregnancy.

Randomly selected progeny from each treatment group were bred as
adults, and allowed to litter. Results of this breeding are presented
in Table 6. No differences in mean gestation length, nor litter size
were detected except in the group prenatally stressed from days l8—21.
While gestation length is significantly less (p<.001) for treated
versus control females, the difference is magnified by the precise
littering of the prenatally stressed group. Certainly, prenatal
stress did not act to lengthen gestation in any circumstance.

These females were permitted to suckle their offspring for 14
days to determine lactational sufficiency. While no attempt was made
to balance the litter size, only one control litter and one treated
litter were not maintained. Pups were at comparable weight when

measured at birth, 7, and 14 days of age (Table 6).

31

Hoo.va
««

mo.va
K.

.wcnom o>HH nuHs woumaHanmu amen moHoamcwonm omonu mo sumaoH COHumumou

n

.m.m H ooozo

 

 

 

 

 

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mo.Hn o.o mH.Hnwm.on ««w~.nom.mn mH.an.N~ N o on anion
mn.nn o.HH mo. Hom.on om.nno.mm owo.nno.- so o m mHuon
mm u mm u mm u we Honoov
Hm>noueH
oNHmnouuHH o>HH nuwdoH aoHumumoo unon< * udenumom

a

 

noooomoom no Hnuwn no .nnuon .mnuon anon so one oomnuoomn ooo
one connuoomo aoou annoa monan oonooononao< moonom oononoooe no ooooom

m MHm<H

Ho.va
«

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oomHIOOMH new on: OOHHIoomo aonm zHHmw ooHBu wommmnum uaHmnumon ono3 mumn unmawonmo

.vommonum hHHmumcmnm n m “Honucoo u U “mGOHumH>onnn<v
.msm non Aawv uano3 owmnm><o

.wcsoh o>HH £DH3 woumaflanou 30H£3 mmHodmcmonm omoeu mo numcoH coHumumou

 

32

 

 

n
.m.m H Goose

mo.- mo.MH m.m mm. H n.0H soc.“ o.NN w

om.m~ mw.mH Nw.m we. Haw.m mo.“ m.N~ m HNImH

oq.HN mo.NH Ho.o mo.HnmN.0H mH.HmN.NN w

mm.- om.MH mo.m om. n n.OH mo.nm~.- OH mHIeH

ac.HN mm.NH m~.o Nu. HMH.OH co.“ o.NN m m

wo.H~ mq.NH oom.m mom. HNN.HH who.HHH.NN OH mo MHIOH

whom «H mmmn n nunHm oNHmnouuHH w Hm>noucH

o>HH nan aoH GOHumummo : unmaumona

Aawv u: nouuHH Hmumaonm

 

mumm onawm vomwonum hHHmumaonm mo nquanomm mam huHHHunom

o MHm<H

33
Discussion

The nocturnal surge of prolactin during early pregnancy is
thought to be necessary for the maintenance of the corpus luteum of
pregnancy (Smith 25 31., 1975). The results of the first experiment
demonstrated that stress was able to interfere with the nocturnal
surge of prolactin, however, no changes were detected in the frequency
of abortion, gestation length or size of litters in female rats sub-
jected to 4 days of stress treatment during early pregnancy. These
results suggest that either the large nocturnal surge of prolactin is
not necessary to maintain a successful pregnancy, or that the surge,
although interrupted, may have been delayed in appearance for the
duration of the stress. Additionally, the stress did not delay nor
decrease implantation, as the length of gestation was not influenced
by the 4 day stress regime, nor was the litter size affected.

These results are in agreement with those of Castro-Vazquez 35
El, (1975), in that immobilization did not affect the number of
embryos that successfully implanted. In that study, however, no
attempt was made to assess the normalcy of the offspring, as the
mothers were sacrificed on day 6 of pregnancy. In the present study,
when litters were pared to 3 males and 3 females each, no difference
in litter weight was detected throughout the 28 day postnatal measure-
ment period. These data indicate that although the mother was sub-
jected to a stresser of sufficient intensity to alter pituitary
hormonal support of pregnancy during the stress treatment, it was not
sufficient to cause adverse effect, on the pre- or postnatal growth of

her offspring.

34

Earlier studies have demonstrated increased abortion and fetal
malformation when restraint stress was administered for several days
(Euker and Riegle, 1973).

In the second study of this experimental sequence, restraint
stress treatment in the afternoon did not appear to alter the occurr-
ence or magnitude of the nocturnal prolactin surge following one or
four days of treatment. Thus, it would appear that the nocturnal
surge is relatively insensitive to stress administered earlier in the
day. This phenomenon may explain why Freeman §£_§l, (1974) suggested
that the nocturnal surge was insensitive to stress as surgical and
handling procedures in their study were performed during the daylight
hours.

Although restraint stress treatment administered in the afternoon
on four consecutive days during midpregnancy tended to increase the
number of litters that aborted, the change was not significant. In
agreement with Euker and Riegle (1973), the effect of stress on litter
size was all or none, as no difference was measured in litter size
among those females that successfully delivered live young. Gestation
length was not altered by restraint stress administered on days 4-7,
8-11, or 12-15 of pregnancy. These data are also consistent with
those of Barlow g; 31. (1978) in that restraint stress during mid-
pregnancy did not affect gestation length.

In this experiment, litters were not culled to balance litter
effects, however, at day 21 following parturition, the pups were
weaned, and only one or two pups per litter were saved for develop-

mental studies. The date of vaginal opening, which signified the

35
first estrus, was not altered by prenatal stress treatment. When
these same prenatally stressed females were bred to control males, no
effect of maternal stress was detected on ovarian cycles or fertility
as each female produced a healthy litter of pups.

In the third experiment, the restraint interval was altered such
that pregnant females were subjected to two hours of restraint twice
daily. The four day treatment periods were chosen to reflect such
times during which specific developmental processes were occurring.
Differentiation of the gonads begins at mid-pregnancy in the rat, and
testosterone is first measurable in fetal males around day 14 of
pregnancy (Gladue, 1979). Peak testosterone concentrations in the
fetal and maternal circulation are found around day 18-19 of pregnancy
(Weisz and Ward, 1980).

An increased incidence of abortions in rats stressed from days
lO-l3 of pregnancy, again affected litter size in an "all or none"
fashion. Non-aborted pregnancies exhibited normal gestation length
and litter size. During the latter half of pregnancy, i.e., days 11-
22, the corpora lutea are no longer dependent upon the pituitary for
support (Pencharz and Long, 1942). Instead, C.L. function is main-
tained by rat placental luteotrophin (rPL). At present, the effects of
stress on placental luteotrophin are unknown. Thus, the increased
abortion shown with this treatment may be due to altered concentra-
tions of rPL or perhaps due to some non-endocrinological phenomenon
such as decreased uterine blood flow.

While the incidence of abortion was not influenced by restraint

stress administered on days 14-17 of pregnancy, the gestation length

36
was significantly increased. The increased gestation length experi—
enced by the restraint stressed females may have contributed to the
higher number of litters that included 1 or more stillborn pups.

Restraint stress administered on days 18-21 of pregnancy had no
effect or incidence of abortion, length of gestation or live litter
size. These data suggest that stages of pregnancy are differentially
sensitive to stress, and that a stressor of sufficient intensity
administered in the latter half of pregnancy can interrupt or delay
the reproductive process. In that regard, our results are in agree-
ment with those of Barlow 23 a1. (1978). In contrast to the study of
Barlow_g£ pl. (1978), we did not find that restraint stress admini—
stered on days 18-21 resulted in a shortened gestation period or
increased perinatal death.

In the present study, there were no significant differences in
the reproductive performance between prenatally stressed and control
females except that when mated, the 18-21 prenatally stressed group
delivered their litters significantly sooner than controls. In all
cases, prenatally stressed females were able to successfully nurse
pups when measured at 7 and 14 days following parturition. This
finding is in direct conflict with the work of Herrenkohl (1979) and
Herrenkohl and Gala (1979), who claim that prenatally stressed female
rats are less fertile and unable to sustain lactation past 10 days
following parturition.

This discrepancy may be due in part to the different nature of
the stress employed, or possibly due to differences in the strain of

rats. However, there is another possible explanation of the

 

37
conflicting results. Herrenkohl does not mention the incidence of
abortion which followed the restraint stress, nor does she indicate
the source of prenatally stressed female offspring. Chapman and Stern
(1978) have conducted behavioral studies using an identical stressor
to that employed by Herrenkohl, and have found that all treatment
effects are eliminated if variation due to litter effects are removed.
Therefore, if an investigator was not careful to either balance
litters throughout treatments or control for such effects by random
selection as was done in the present study, the influence of an indi-
vidual litter may overwhelm the results of a particular stress treat-
ment. In the behavioral studies cited, one might expect to find a
difference in masculinity or femininity if offspring were from litters
dominated by one sex or the other (Clemens g; 31., 1976). This is a
problem inherent in the use of polytocous animals for such studies.

In the present investigation, attempts were made to minimize
litter effects by utilizing not more than one or two female litter
mates whenever possible, in subsequent experimentation. In all cases,
adult female rats that were stressed prenatally demonstrated complete
reproductive competence when compared with non-stressed control
animals. While laboratory conditions and strain differences may
explain significant findings in other laboratories, these results
suggest that investigators must guard against litter effects contri-

buting a major source of variation to the results.

CHAPTER 3
Prolactin Release and Tuberoinfundibular Dopaminergic
Nerve Activity During Pregnancy and Pseudopregnancy

During pseudopregnancy and early pregnancy in the rat, the pat-
tern of prolactin release is characterized by two daily surges, one
diurnal and the other nocturnal (Butcher g£_§l,, 1972; Freeman g£_al,,
1974). Both surges are thought to be required for the initiation and
maintenance of luteal function in the intact rat (Smith 35 31., 1975).
In Chapter 2, we reported that stress was able to interfere with
either surge, yet in those same studies we were not able to demonstrate
an increased incidence of abortion during early pregnancy when the
prolactin surges are manifest. Due to this somewhat unexpected
finding, we set out to more fully characterize the release of prolac-
tin during pregnancy and pseudopregnancy and to explore in more
detail, the effects of restraint stress on the surge release of
prolactin.

Very little is known about the neural mechanisms that regulate
the surges or the effects of stress on prolactin secretion during
pregnancy or pseudopregnancy. A variety of noxious stimuli such as
aortic cannulation, sham surgery, and serial blood sampling have been
shown to temporarily suppress the diurnal, but not the nocturnal
prolactin surge of pseudopregnant rats (Freeman g£_al,, 1974). A
similar effect on the diurnal surge of pregnancy has also been re-

ported (Riegle and Meites, 1976b). Whether these changes are mediated

38

39
by decreased release of a prolactin releasing factor (PRF) or the
increased action of an inhibitory factor (PIF) is unknown. As an
additional aspect of the present study, we studied the activity of the
tuberoinfundibular (TI) dopaminergic neurons that are thought by many
to be involved in the regulation of pituitary prolactin and LH re—

lease.

Prolactin, Dopamine and Tuberoinfundibular Dopaminergic Neurons
Studies that have demonstrated a tonically negative influence of
the hypothalamus over prolactin release date back to 1954 (Everett,
1954). Since that time, the quest for hypothalamic factors that
either inhibit or promote the release of prolactin from the anterior
pituitary, has generated many studies, and their results have been
covered by several thorough reviews (Meites g£_a1,, 1972; MacLeod,
1976; Fernstrom and wurtman, 1977). Dopamine (DA), and pharmacologi-
cal agonists of DA were demonstrated to have potent inhibitory effects
on PRL secretion, iplyipgp_and 1p Kipp. Dopamine is thought to act
directly on the adenohypophysis, as physiological concentrations of DA
suppressed the release of prolactin from cultured pituitaries (Shaar
and Clemens, 1974). Hypothalamic extracts stripped of catecholamine
content by alumina absorption lost their ability to inhibit PRL
release from identically cultured pituitaries (Shaar and Clemens,
1974). The absence of DA receptors in the medial basal hypothalamus
and the presence of such receptors in the pituitary further suggested
that hypothalamic DA must be released and transported to exert its

effects directly on the pituitary (Brown 35 31., 1976).

40

The source of the pituitary prolactin inhibiting dopamine is
thought to be the tuberoinfundibular dopaminergic (TIDA) neurons,
whose cell bodies lie in the periventricular-arcuate nuclei of the
hypothalamus. In the median eminence the TIDA terminals juxtapose the
‘median eminence capillary plexus of the hypothalamo-hypophyseal portal
vessels that course down the pituitary stalk, and are the major vascu-
lar supply for the pituitary (Figure 4) (Renaud 3£_31,, 1978; Fuxe 33
31,, 1976). Thus, the TIDA neurons may exert major effects on the

pituitary by the release of dopamine directly into the hypophyseal

portal vessels.

Dop3mine in the Portal Vasculature

 

The actual measurement of dopamine in hypophyseal portal plasma
of the rat at concentrations higher than peripheral plasma (Ben—
Jonathan 3£_31,, 1977) and the demonstration that DA, at portal blood
concentrations, was able to suppress prolactin release 1p_!1!3_also
suggest that dopamine is an endogenous PIF (Gibbs and Neill, 1978).

Compatible studies have shown that pharmacological manipulation
of dopaminergic neurotransmission resulted in an inverse relationship
between serum prolactin and hypophyseal portal plasma concentrations
of DA (Horowski and Graf, 1976; Gudelsky and Porter, 1979). However,
attempts to simultaneously measure physiological changes of peripheral
PRL and portal DA in the same animal have failed to demonstrate such a
"mirror image" relationship (DeGreef and Neill, 1979).

Despite the failure to prove the reciprocal model of the DA-PRL

release, recent work has further demonstrated association between the

41

two substances within the pituitary. Approximately 75% of all pitui—
tary DA is internalized specifically within prolactin granules of
lactotrophs, while no such connection appears to exist for the control
of LH, growth hormone, follicle stimulating hormone, adrenocortico-
trophic hormone or thyroid stimulating hormone (Nansel 33 31, 1979).
During the estrous cycle, pituitary DA content was recently found to
vary inversely with peripheral prolactin concentrations (Chiocchio 33
31,, 1980). These results suggest a functional relationship between

anterior pituitary dopamine content and pituitary prolactin release.

Hormonal Effects on TIDA Neuronal Activity

 

In the attempt to understand the neuronal-hormonal control of
prolactin release, it is necessary to characterize changes in neuronal
activity with alterations of physiological states. Histofluorescence
techniques demonstrate that castration as well as ovarian steroid
supplementation can alter the activity of TIDA neurons (Fuxe 3£_31,,
1969). When specific anterior pituitary hormones were injected into
rats, it was found that PRL, but not LH, FSH or ACTH caused an in-
crease in TIDA neuronal activity. These results suggested that PRL
may act, in part, as its own regulator via a feedback system from the
pituitary to the TIDA neurons. These findings were substantiated when
it was shown by a sensitive radioenzymatic assay that PRL induced a
specific activation of TIDA neurons 10-26 hours following treatment in
OVX rats (Gudelsky 3£_31,, 1976), and induced an increase of DA into
hypophyseal portal blood in male rats (Gudelsky and Porter, 1980).

When estimates of TIDA neuronal activity were obtained by the

measurement of the accumulation of DOPA after the administration of a

42

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43

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44
decarboxylase inhibitor (Demarest and Moore, 1980) the results follow-
ing pharmacological and endocrinological manipulations were similar to
those in the aforementioned studies in that: l) the administration of
haloperidol, a dopaminergic antagonist known to increase serum PRL
concentrations, resulted in an increase of TIDA neuronal activity 16
hours following treatment, and 2) the estrogen-induced increase in
neuronal activity within the median eminence was abolished in the
hypophysectomized rat, which suggested that the effect of the steroid
was due to its facilitory action on pituitary prolactin release.

The exact mechanism by which prolactin causes the latent activa-
tion of DA synthesis within the terminals of tuberoinfundibular
neurons is not known. Recent studies have shown that prolactin in-
duced a delayed increase in the activity of tyrosine hydroxylase
selectively within the median eminence (Nicholson 33 31., 1980).

Since tyrosine hydroxylase is the rate limiting enzyme in the synthe-
sis of DA (see Figure 5), it is possible that the latent period may
reflect the time required to synthesize and transport new enzyme to
the TIDA nerve terminals within the median eminence. Consistent with
this hypothesis is the finding that cycloheximide, a protein synthesis
inhibitor, administered up to 4 hours following icv PRL treatment
blocked the expected increase of TIDA neuronal activity (Johnston 33

§_1_., 1980).

Stress and Biogenic Amines

 

The effect of stress on pituitary hormone release could also in-
volve alterations in hypothalamic hypophysiotropic factors. Stress-

induced changes of catecholamine content have been shown within the

45

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47

whole brain (Moore and Lariviere, 1964; Corrodi 33 31., 1968) and the
hypothalamus (Palkovits 3£_31,, 1975; Keim and Sigg, 1976). The
intensity, duration, and type of stressor affect the magnitude of the
catecholaminergic response (Keim and Sigg, 1976) as does the repro-
ductive status (e.g., pregnant) of the animal (Moyer 3£_31,, 1977).

There is general agreement that hypothalamic monoamine neurons
exert an influence on the release of gonadotropins (Kordon and Glo-
winski, 1972; Fuxe. E 31,, 1976). However, results of early attempts
to correlate stress-induced changes in pituitary and peripheral hor-
mone release with whole brain concentrations of dopamine were not
consistent (Carr 35 31., 1968; Corrodi 33 31., 1971). The development
of sensitive assay techniques permitted the measurement of catechol-
amines in discrete hypothalamic nuclei (Kventnansky 3£_31,, 1977) that
may be associated in the stress-induced changes of hormone release.

The study of acute stress revealed that at least two neuronal
systems were operational in the control of prolactin release. The
initial rise in serum prolactin concentrations was not due to the
inhibition of dopamine, but to enhanced activity of such substances as
norepinephrine, serotonin, or TRH (Blake, 1974; Marchlewska—Koj and
Kralich, 1975; Collu 33 31., 1979; Mueller 3£_31,, 1976). The admi-
nistration of DA agonists blocked the stress-induced increase or
prolactin, but treatment with DA antagonists was permissive to in-
creased prolactin secretion (Meltzer 3£_31,, 1976).

Immobilization stress produced a biphasic effect on serum prolac—
tin levels (Riegle and Meites, 1976b; Tache 33 31., 1976). At the
onset of stress, prolactin release is stimulated. If the stress is

continued beyond one hour, prolactin release declines to very low

48
levels. Concentrations of another hormone commonly associated with
stress, corticosterone, were increased throughout the stress interval,
and remained elevated when the stress was administered over a period
of days (Riegle, 1973; Tache 33 31,, 1979; Kawakami 33 31., 1979),
suggesting that PRL and ACTH secretion are controlled by different
neuroendocrine mechanisms.

The administration of TRH or noxious stimuli following six hours
of restraint in male rats had no effect on stress depressed serum PRL
concentrations (Kawakami 33 31., 1979). The injection of a dopamine
receptor blocker resulted in the elevation of prolactin levels to that
of controls. These results indicated that a dopaminergic mechanism
was involved in the depressed serum concentrations of prolactin which
followed a lengthy 6 hour stress.

Unlike the chronically stressed rat, the lactating rat is charac-
terized by sustained high levels of prolactin and low concentrations
of corticosterone (Stern and Voogt, 1973/74). Serum prolactin concen-
trations are decreased when the lactating female is pup-deprived. A
variety of stressors can induce a modest increase in serum.prolactin
levels following pup removal. These stress-induced increases of
prolactin last for less than 60 minutes in the pup-deprived rat. The
return of the pups and normal suckling results in greatly elevated and
sustained prolactin values. Grosvenor has hypothesized a multi-phase
process in which prolactin is released from the pituitary following
various neurogenic stimuli (e.g., suckling) that transform the storage
pools of PRL into a releasable form (Grosvenor 33 31., 1979). In the

normal animal, the releasable PRL pool is small, and TRH and/or ether

49
stress cause only a brief (<60 min) increase in serum PRL concentra—
tions. Once the pituitary prolactin content is transformed by the
suckling stimulus, TRH or ether stress can sustain elevated PRL
concentrations for extended periods of time (Grosvenor 33 31., 1979;
Grosvenor 35_31,, 1980). The "depletion-transformation" process
appears to be regulated by DA, as 5-bromo-a-ergocryptine (a dopamine
agonist) administered before but not after suckling, prevented deple-
tion of PRL (Grosvenor 33 31., 1980). These results suggest that
pituitary DA is a major inhibitor of sustained elevations in PRL
release, and are consistent with the depressed TIDA activity and
decreased anterior pituitary DA content seen during suckling and
lactation (McKay 33 31., 1980). While it is generally agreed that DA
released from TIDA neurons tonically inhibits the release of prolac-
tin, very little is known about the activity of these neurons during
pregnancy. The demonstration of elevated concentrations of DA in the
hypophyseal portal blood during pregnancy and parts of the estrous
cycle was necessary to establish tuberoinfundibular DA as a major PIF
(Ben-Jonathan 33 31., 1977), but procedural problems such as the
depressive effect of anesthesia on PRL release and the time and stress
involved in obtaining a blood sample preclude the usefulness of this
technique to establish the exact relationship between TIDA neuronal
activity and pituitary PRL release during pregnancy and pseudopreg—
nancy (Ben-Jonathan 33 31., 1977; DeGreef and Neill, 1979). The
objectives of the present study include characterization of TIDA
neuronal activity during normal pregnancy and pseudopregnancy in the
rat by determining within the median eminence the rate of DOPA accu-

mulation following the inhibition of DOPA decarboxylase. This

50
technique has been shown to be a reliable technique to estimate TIDA
neuronal activity (Demarest and Moore, 1980). If tuberoinfundibular
DA is a hypophysiotropic hormone with PIF capabilities, it is reason-
able to suspect that the surge release of pituitary prolactin may be
accompanied by alterations in TIDA neuronal activity.

In the previous chapter, it was shown that restraint stress
interfered with the diurnal and nocturnal surge release of PRL during
early pregnancy. A second objective of this thesis will be to examine
more fully the effects of a 4 hr restraint stress on the release of
prolactin, and to measure related changes in DOPA accumulation. In
the previously described studies, it could not be determined whether
the surge release of PRL was blocked or delayed by restraint stress.
In this study, the release of PRL was characterized for over 24 hours
after the initiation of restraint.

If a functional feedback loop is operational between the surge
release of prolactin during pregnancy and pseudopregnancy and TIDA
neuronal activity, then the stress-induced alteration of PRL should be
reflected in changes in the accumulation of DOPA within the median
eminence.

A final study will examine the feedback hypothesis in pseudopreg-
nant animals that have been ovariectomized. The diurnal surge of PRL
is absent in the OVX cervically stimulated rat (Freeman 3£_31,, 1974);
thus, any PRL feedback effects on TIDA neurons would be attributable

to the slightly attenuated nocturnal surge.

51
Materials and Methods

Animals - Housing and Handling

 

The animals were caged and maintained under the same conditions
as those described in Chapter 2. In studies that required pregnant
rats, the breeding was conducted as previously described. Pregnancy
was confirmed during necropsy by the presence of implantation sites.
Pseudopregnancy was induced by electrical stimulation of the cervix
with a probe (10 sec with 240 pulses/sec and a pulse duration of 1
msec at 25 volts) at 0930 hr and 1630 hr estrus and at 0930 diestrus.
The first day of cervical stimulation was considered day 1 of pseudo-
pregnancy. Pseudopregnancy was confirmed by the observation of leuco-
cytic vaginal cytology for six days. In those cervically stimulated
animals subjected to ovariectomy, vaginal cytology was no longer a
useful indicator of the successful induction or maintenance of pseudo-
pregnancy. Instead the presence of the 0300 hr prolactin surge
(Y :_E - l S.D.) on Day 5 served as an index of successful cervical
stimulation.

Ovariectomy was performed under ether anesthesia via a mid ventral
incision. Both uterine horns were exteriorized through a 1 cm incision
and the ovaries were teased away from the uterus and connective tissue.
After the uterus was repositioned, the peritoneum was restored with
gut suture, and the exterior was closed by stainless steel clips.

Sham ovariectomies for the control group followed the same procedure
except that ovaries remained intact.

Restraint stress as described in Chapter 2 was administered to

groups at intervals outlined in Results.

52

Tissue Handling and Assay - DOPA Accumulation

 

The 13321!3_rate of DA synthesis was estimated by measuring the
rate of DOPA accumulation following the administration of a decar-
boxylase blocker (Figure 5). Each rat was injected with 3-hydroxy-
benzylhydrazine (NSD 1015, 100 mg/kg i.p.; Sigma Chemical Co., St.
Louis, MO) 30 minutes prior to decapitation. The brains were quickly
removed from the skull and placed on a cold plate. The median emi-
nence was removed with the aid of a dissecting microscope. The
striatum, olfactory tubercle, nucleus accumbens and posterior pitui-
tary were dissected by published experimental techniques (Demarest 35
31,, 1980; Glowinski and Iverson, 1966; Horn 33 31., 1974). The
median eminence and posterior pituitary were homogenized in 20 ul,
and the striatum, olfactory tubercles and nucleus accumbens were
homogenized in approximately 10 volumes of cold 0.2 N perchloric acid
containing EGTA (10 mg/100 ml). Following centrifugation 10 m1 of the
supernatant from individual tissue samples were assayed for DOPA
assays by radioenzymatic procedure (Demarest and Moore, 1980) and the
tissue pellets were analyzed for protein by the method of Lowry (Lowry
3£_31,, 1951). DOPA concentrations were expressed as ng DOPA/mg
protein. The preceding technique is performed routinely in the
laboratory of Dr. K.E. Moore, Department of Pharmacology and Toxi-
cology, Michigan State University, East Lansing, MI. All assays
reported in this report were performed in that laboratory under the
direct supervision of Dr. K.T. Demarest, Departments of Pharmacology

and Toxicology, and Physiology, Michigan State University.

53

DA Steady—State Concentration

 

DA content of the anterior pituitary was measured in rats by a
radioenzymatic technique as described (Umezu and Moore, 1979). Imme-
diately following sacrifice by decapitation, the brains were removed
to expose the pituitary. The bi-lobed anterior pituitary was sepa—
rated from the neuro-intermediate lobe and immediately homogenized in
30 ul of 0.2 N perchloric acid containing EGTA (10 mg/100 ml).
Protein was measured as previously stated and the data was expressed

in ng DA/mg protein.

Blood Collection

 

Blood samples were collected following decapitation or by orbital
sinus puncture as specified in Results. Radioimmunoassay for PRL

and LH was conducted as described in Chapter 2.

Statistical Analysis

 

Initial studies designed to characterize PRL, LH, and DOPA accu-
mulation during pregnancy, pseudopregnancy, and diestrus were analyzed
by computer using a one-way analysis of variance. Between group
comparisons were made by the Student-Newman-Keuls' test (Nie 3£_31,,
1975). Only differences with a probability of error less than 5% were
considered significant. Prolactin data were transformed to natural
log values to provide homogeneity of variances.

Further studies involving restraint stress, bleeding stress and
ovariectomy were analyzed by computer using the two-way analysis of
variance program Balanova (Coyle and Frankmann, 1980). The prolactin

data were subjected to a natural log transformation to achieve

54
homogeneity of variance. Selected 3 priori comparisons were made
using the Student's pftest based upon the mean square error (within)
from the two-way analysis of variance (Sokal and Rohlf, 1969; Rohlf

and Sokal, 1969).

Results

Characterization of Tuberoinfundibular Dopaminergic Neuronal Activity
During the Daily Surges of Prolactin Secretion in Pregnant and
Pseudoprggnant Rats

 

Serum prolactin and LH concentrations were analyzed at specified
times on day 6 of pregnancy and pseudopregnancy and on the second day
of diestrus. To avoid a possible increase in prolactin secretion
related to stress associated with the handling and injecting of the
animals, blood samples were collected from pregnant and pseudopregnant
animals 1 day prior to the determination of DOPA accumulation and from
diestrous animals on the corresponding day of the cycle preceding the
experiment. Thus, on day 6 of pregnancy or pseudopregnancy the rats
were randomly subdivided into 8 groups of 8 rats each. A single blood
sample (approximately 1 ml) was collected from each rat by orbital
sinus puncture under light ether anesthesia at one of the following
times: 0300, 0600, 0900, 1200, 1500, 1800, 2100, and 2400 hr. Rats
included in the diestrous study were similarly divided into 8 groups
of 8 animals each and subjected to a blood collection at one of the
same time points. During the dark period all manipulations were
carried out under a red light.

During day 6 of pregnancy, two peaks of prolactin were detected;

a nocturnal surge which peaked at 0300 hr and a diurnal surge which

55
peaked at 1800 hr, just prior to the dark period (Figure 6). On day 6
of pseudopregnancy, the pattern of prolactin secretion was similar to
that observed during pregnancy; a nocturnal peak was detected at 0300-
0600 hr and a diurnal peak at 1800 hr (Figure 7).

There was also a cyclic variation in the rate of DOPA accumula-
tion in the median eminence of the pregnant and pseudopregnant rats.
In the pregnant rats there were two daily low points in the rate of
DOPA accumulation with the nadirs at 0600 and 2100 hr (Figure 9). In
the pseudopregnant rats the nadirs occurred at 0300 and 1800 hr
(Figure 10). On the second day of diestrus the serum concentrations
of prolactin and the rate of accumulation of DOPA in the median emi-
nence did not change throughout the 24 hr period (Figure 8 and 11).
While there were no significant changes in the serum concentrations of
LH over the 24 hr period in pregnant, or diestrous animals (Figures 6
and 8, the pseudopregnant group exhibited a slight but significant
change with peak values at 1200 and 1500 hr and a nadir at 0600 hr.

Changes in DOPA accumulation throughout the 24 hr period of
pregnant and pseudopregnant rats were restricted to the median emi-
nence; there were no consistent changes in DOPA accumulation in any
other brain regions (Tables 7 and 8).

A comparison of the rates of DOPA accumulation in the median
eminence observed in pregnant, pseudopregnant, and diestrous rats
(Figures 9, 10 and 11) might suggest differences in DA synthesis rates
during these different reproductive states. Because of a number of
variables (e.g., between radioenzymatic assays, time of year, etc.) it
is not possible to make direct comparisons between the results ob-

tained in these different studies. Accordingly, an additional study

56

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70
was undertaken to compare the rate of DOPA accumulation in selected
brain regions on day 7 of pregnancy and pseudopregnancy and on the
second day of diestrus. All animals were sacrificed between 1140 and
1220 hr, a time of maximal rates of DOPA accumulation in the pregnant
and pseudopregnant rats. Samples of each brain region were analyzed
in a single assay so that a direct comparison could be made between
the 3 groups of animals. Rates of DOPA accumulation in the median
eminence of pregnant and pseudopregnant rats were similar and both
were significantly greater than that in diestrous rats (Table 9).
There were no differences in the rates of accumulation of DOPA in any

other brain regions.

Effects of Blood Sampling on Prolactin Durinngregnancy

Serum prolactin concentrations were measured at specified times
on day 7 of pregnancy. In the previous experiment, animals were sub—
jected to blood sampling on day 6 of pregnancy and DOPA accumulation
determination in the same rat 24 hours later. To assess if blood
sampling and handling on day 6 of pregnancy affected serum PRL concen-
trations 24 hours later, blood samples were collected from animals on
day 6 of pregnancy and again 24 hours later and in control rats sub-
jected to blood sampling on day 7 only. Thus, on day 6 of pregnancy,
half of the rats were randomly subdivided into 8 groups of 6 rats
each. A single blood sample (approximately 1.0 ml) was collected from
each rat via orbital sinus puncture under light ether anesthesia at
one of the following times: 0300, 0600, 0900, 1200, 1500, 1800, 2100
and 2400 hr. Twenty-four hours later the same rat was again subjected

to orbital sinus puncture. The remaining rats were similarly divided

 

71

TABLE 9

DOPA Accumulation at 1200 hr in Pregnant, Pseudopregnant
and Diestrous Female Rats

 

 

Pregnant Pseudopregnant Diestrous
Median Eminence 17. 6:138 18. 1:1.14a 11.510. 8
Corpus Striatum 9.0i0.5 8.4i0.4 8.2il.2
Nucleus Accumbens 10.1i0.9 9.9i0.3 9.4i0.7
Olfactory Tubercle 7.4iO.4 7.8i0.5 7.6i0.4
Posterior Pituitary 0.59:0.04 0.49:0.03 0.60i0.06

 

Pregnant (Day 7), pseudopregnant (Day 7) and diestrous rats re-
ceived NSD 1015 (100 mg/kg, i.p.) 30 min prior to sacrifice at
1200 hr i 20 min. DOPA concentrations are expressed as ng/mg
protein, n = 8—11.

aValues that are significantly different from values in correspond-
ing brain regions of diestrous rats (p<0.05).

 

72
into 8 groups of 6 animals, but were subjected to orbital sinus punc-
ture at one at the same time points on day 7 only. As in the previous
experiment, all manipulations of the rats during the interval when the
colony lights were out were carried out under a red incandescant
light.

Two peaks of prolactin were measured in both treatment groups
with a nocturnal surge that peaked at 0300 hrs and a diurnal surge
that peaked at 1800 hrs (Figure 12). The concentrations of serum
prolactin were very similar throughout the twenty—four hour period,
however, a statistically significant difference (p<.05) occurred at
2100 hours.

The Effect of an Acute 4 hr Restraint Stress Administered During the

Nocturnal or Diurnal Surge of Prolactin and DOPA Accumulation in
Pregnant and Pseudopregnant Rats

 

 

 

Pregnant and pseudopregnant rats were subjected to a 4 hr restraint
stress administered during the expected nocturnal or diurnal surge of
prolactin. In this preliminary experiment, serum prolactin and LH
concentrations were analyzed in samples collected immediately preceding
the restraint period and at the end of the 4 hr treatment. Thus, on
day 5 of pregnancy or pseudopregnancy, rats were randomly subdivided
into 4 groups of 8 rats each. At 2300 hrs on day 5, 2 groups of rats
were subjected to orbital sinus puncture under light ether anesthesia.
One group was then restrained until 0300 hrs day 6, while animals in
the second group were returned to their home cages and remained un-
disturbed during the four hour interval. Another blood sample was
collected from the same animals at 0300 hr on day 6. Both groups were

then returned to their respective cages until 1200 hrs day 6 when they

73

.Honakm onu mo moHomn onu swan mmoH mH mm man

csocm mH moaHH on onoaz .Mm H mueomonmon ucHoa some swoonSD oeHH HmoHuno> man
mam maOHumeHEnonoo e no some men mucomonaon namnw man no uaHom 30mm .nHao n mom
:0 onn onoB none mnmn nemamonm uaomonaon moHonHo womoHo osu mam .noHHnmo on: «N
unauocse msch Hmanno ou monoanSm coon mm: umau mHmchm omosu ucomonaon moHonHo
ammo use .moamewmnm mo m zoo no mHm>noucH noon m um onsuoeom msch Hmanno

mH> wouooHHoo mmHmamm voOHn GH woeHenouov onus :HuomHonm mo chHumnuaoocoo ennom

.%ocmcwonm
anmo wcHnso meHumnueoocoo cHuomHonm adnmm co onouuesm moeHm Hmanno mo uoommm

.NH onownn

 

74

 

 

 

 

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75
were sacrificed for DOPA accumulation determination. The remaining
two groups were undisturbed until 1430 hrs day 6 when they were
likewise subjected to orbital sinus puncture. From 1430-1830 hrs one
group was restraint stressed, while the other animals rested in their
home cages. At 1830 hrs, animals of both remaining groups were again
handled for blood collection, and were returned to their cages until
determination of DOPA accumulation at 2400 hrs..

Restraint stress from 2300 hrs of day 5 of pregnancy until 0300
hrs on day 6 interfered with the appearance of the nocturnal surge of
PRL (p<.001) (Figure 13). A similar result was observed in pseudo-
pregnant rats (Figure 14). There were no significant differences due
to restraint stress in the rate of DOPA accumulation within the median
eminence when measured at either 1200 hrs day 6 of pregnancy or pseudo-
pregnancy (Figures 13 and 14).

The diurnal surge of PRL was absent at 1830 hr in pregnant and
pseudopregnant rats restraint stressed from 1430-1830 hr on day 6
(Figures 15 and 16). No significant changes in the rate of DOPA
accumulation within the median eminence were measured at 2400 hrs in
either pregnant or pseudopregnant rats (Figures 15 and 16). Serum LH
concentrations were not altered by restraint stress in either pregnant
or pseudopregnant rats.

The Effect of Restraint Stress During the Nocturnal Prolactin Surge
of Pregnancy on the Pattern of Prolactin Release

 

 

Serum prolactin concentrations were analyzed at specified times
on day 6 of pregnancy following a 4 hr restraint stress administered
during the expected nocturnal PRL surge. Although results of the

preliminary experiment demonstrated that restraint stress could

 

76

.Ho.vm n « .Mm H ucomonnon non nomo mono moeHH

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on nOHnn cHa om A.Q.H .mn\wa oonv mHOH sz co>Hooon non noom .aoHumHsasooo
<mon mo neoaonsmooa onu now nn oowH no wooHMHnoom ono3 muon Ammv commonum
ueHonumon woo HOV Honueoo .0 now no nn come no Ho>noueH neoauoonu nn q onu wcH
IsoHHom eHouonoeeH can eoemawonm mo m eon do nn comm no onouoesm odeHm Hoanno
en wouooHHou moHaaom vooHn aH oonSwooE ono3 nHuomHonm mo mGOHumnuaoocoo Banom

.uon unocwona onu :H GOHumHaasoom
<moa new cHuooHonm mo own5m Honnouoon onu co moonum uaHonnmon mo noomwo one

. 3 3&3

PREGNANCY

‘40

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77

DOPA (ng/mg protein)

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.uon uaoewonaoonoma onu GH coHuoHoaoooo
<moo woo eHuooHonm mo ownam Hocnsuoon onu so moonum uaHonumon mo noowwo one

.on onownm

79

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wommonum naHonumon can on Honucoo .eoeoewonm wo o emu do nn ommH no Ho>nouaH
moonum nn H onu nouwo eHouoHooEEH woo nn oqu no onSuocam osch Hoanno

en wouooHHoo oonaom wOOHn :H monomooe ono3 eHuooHone wo mcoHuonueooeoo Bonom

.uon ucoawonm onu aH GOHumHsaaooo
neon cam cHuooHone wo ownsm HocnoHo onn no moonum uaHonumon wo uoowwo one

.mH onownn

PREGNANCY
I430“ I830

81

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82

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.umn newswonmowsomm onu cH COHuoHdaaooo
<moo woo cHuomHone wo ownnm HonnsHo onu no moonum uGHmnumon wo noowwo one

.on ononne

PSEUDOPREGNANCY

83

DOPA (ng/mg protein)

 

 

 

 

 

 

 

 

 

 

 

 

 

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84
interfere with the occurrence of the nocturnal surge of prolactin, it
was not known if the surge was blocked or merely delayed. Therefore,
in the first experiment to be reported here, day 5 pregnant rats were
randomly subdivided into 16 groups of 6 rats each. A 4 hr restraint
stress was administered to 8 groups of rats from 2300 hrs day 5 until
0300 hrs day 6 of pregnancy. The other 8 groups remained in their
respective cages undisturbed during this time interval. A single
blood sample (approximately 1 ml) was collected from each treated and
control rat by orbital sinus puncture under light ether anesthesia at
one of the following times on day 6 of pregnancy: 0300, 0600, 0900,
1200, 1500, 1800, 2100 or 2400.

Restraint stress significantly lowered serum prolactin levels
when measured at 0300 and 0600 hrs (Figure 17). However, the noctur-
nal surge was delayed rather than blocked by restraint stress, as
serum PRL concentrations were significantly elevated over control
levels at 0900 and 1200 hrs. Although the prolactin concentrations of
restraint stressed rats at 1800 hrs (diurnal surge) tended to be lower
than 1800 hr values in non-stressed females, the difference was not
significant.

PRL concentrations at 2100 hrs of restraint stressed rats were
significantly elevated over non-stressed controls, which might suggest
that the 3 hr sampling interval was not adequate to detect a slight

delay in the diurnal PRL peak.

 

85

.mo.vm u « .Honeem onu wo mSHoon onu eonu mon mH mm onu

.e3onm ono moeHH on onon3 new .mm H mucomoneon uaHoe nooo nwsonnu oaHH HmoHuno> one
.ooeHonuoon u monocoo u umcoHuooHenouoo o wo cooe onu mueomonmon nmmnw onu

no ueHom nomm .eococwone wo 0 how no mHo>nouaH nn m um onduocom moeHm Hoanno en
wouooHHoo moHaEmm wooHn aH woeHanouoo onoB cHuooHona wo maOHuonucooaoo Eonom .nn
00mm on wouoHuHeH moonum ucHonumon nn q m on wouoonnsm ono3 moon unocmonm m eon

.uon newswonm
onu :H omooHon aHuooHonm wo anouuoa onu no moonum uaHonumon nn H m wo noowwo one

.nn onownn

86

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87

The Effects of Restraint Stress During_the Nocturnal Prolactin Surge
of Pregnancy on the Rate of DOPA Accumulation

 

 

To further investigate the effects of restraint stress on PRL
release and TIDA neuronal activity during pregnancy, a second experi-
ment was conducted. As in the previous study, 16 groups of 6 pregnant
rats each were evenly divided between restraint stress and control
treatments. In this experiment 2 blood samples were collected from
each rat. The first at 2300 hrs of day 5 and the second at 0300 hr on
day 6 of pregnancy. The rats designated as the treatment group were
subjected to restraint stress between those blood collection times,
while the remaining control rats were returned to their home cages.
The rate of DOPA accumulation within the median eminence was measured
in each rat at one of the specified times on Day 6 of pregnancy as
listed in the previous study. Only those restraint stressed rats in
which the 0300 hr PRL surge was interfered with were included in the
results.

The rate of DOPA accumulation within the median eminence was
significantly elevated from non-restrained controls at 0600, 1800 and
2400 hrs (Figure 18). In accordance with the preliminary study, no
significance between group difference in the rate of DOPA accumulation
in the median eminence was observed at 1200 hrs. The significantly
elevated rates of DOPA accumulation measured within the median emi-
nence of restraint stressed rats at 1800 hrs are consistent with the

restraint stress induced delay of prolactin release.

 

88

.nnnoooo Hooononooo now an onomnn on ooowon oom .H.o.n .wx\wa oonv anon

mmz wo eonuoonnH no vo>Hooon HoeHeo nooo .oonHnoom ou noHnm .eoeonwonm wo o

emu co mHo>nouaH nn m no voonHnoom moon cH ooeHenouov mos eoHuoHoeoooo «mom .nn
comm no wouoHuHaH moonum ucHonumon nn H m on wouoomnsm ono3 moon ucoemone m eon

.uon newswonm onu wo ooeonHeo conoE
onu CH GOHuoHseoooo <mom wo enouuom onu no moonum uaHonumon nn q o wo uoowwo one

.wn oeomne

89

 

 

 

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TIME (hr)

 

90

The Effect of Restraint Stress During the Nocturnal Prolactin Surge
of Pregp3ncy on the Rate of DOPA Accumulation at Selected Times
After Stress

 

 

 

An additional study was undertaken to examine the rates of DOPA
accumulation within the median eminence of stressed and non-stressed
rats on day 6 of pregnancy. In the previous experiment, the rate of
DOPA accumulation within the median eminence was altered from control
values during the 24 hr period following restraint stress (see Figure
18). Since this was the first study to suggest that restraint stress
alters the activity of TIDA neurons, it was necessary to confirm that
the observed differences were due to stress treatment rather than the
normal variability of TIDA neuronal activity that is detected through-
out the day in pregnant rats. Accordingly, this study was conducted
to compare the rates of DOPA accumulation within the median eminence
of selected times on day 6 of pregnancy following restraint stress
administered from 2300 hrs on day 5 of pregnancy until 0300 hrs on day
6. Restraint stressed females and non-stressed controls were sacri-
ficed for determination of DOPA accumulation at one of the following
times: 0600, 1200, 1800 or 2400. Rates of DOPA accumulation in the
median eminence of rats killed at 0600, 1200, and 1800 hours were
similar to those of the previous study (Figures 18 and 19). However,
when measured at 2400 hrs, DOPA accumulation values between stressed
and non-stressed rats were not significantly different as previously
measured (Figures 18 and 19). These results might suggest that the
2400 hr sampling point falls at a time of transition of PRL release
and TIDA neuronal activity. The large standard error bars depicted in
Figure 17 indicated a large variation in the release of PRL at 2400

hrs. That data indicated that some rats had initiated their nocturnal

 

91

.mo.vnH u « .Mm H

neomonaon non nooo mono mocHH HooHuno> .mGOHuonHanouoo Awmv wommonum uaHmnumon
uano no ADV Honueoo uano wo cooa onu muaomoneon non nuom .nn ooqm no oowH
.oomH .oooo no muon newswonn o moo wo ooconHEo coHuoa onu :H coeHEnouow moB :oHu
IoHnasooo <mon A.Q.H .wn\m8 OOHV mHOH nmz wo SOHDonumHeHer onu wcHonHom .nn
comm no mouoHuHcH mmonum ucHonumon nn q m on wouooflnow onos moon newswone m moo

.moaHu wouooHom no GOHumHsapoom <moa no mmonum uaHonumon nn q m we noowwm

..mH onnwnn

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93
surge of prolactin while others had not. Thus, the discrepancy be—
tween the rates of DOPA accumulation values at 2400 hrs between this
study and the one previous, are consistent with the hypothesis of a
functional relationship between TIDA neuronal activity and pituitary
prolactin release, and may be explained by the number of rats that
had initiated the nocturnal surge of PRL by the 2400 hr sampling
point.

The Effect of Ovariectomy on Prolactin Secretion and Anterior Pituitary
Dopamine Content in Cervically Stimulated Rats

 

 

 

The pattern of prolactin release of cervically stimulated rats
following ovariectomy is characterized by an attenuated nocturnal
surge and the absence of the diurnal surge typical of pseudopregnancy
(Freeman e£_31,, 1974). In this study, serum prolactin concentrations
and steady-state DA concentrations in the anterior pituitary were
measured in cervically stimulated-ovariectomized rats. Eight groups
of 6 rats were ovariectomized on day 2 postcervical stimulation. Sham
surgery was performed on a similar number of animals which served as
the control group. On day 6 postcervical stimulation, each animal was
sacrificed by decapitation at one of the following times: 0300, 0600,
0900, 1200, 1500, 1800, 2100, or 2400 hrs. Trunk blood was collected
from each rat, and the anterior pituitary was quickly removed from the
brain for measurement of DA concentration. The concentrations of
serum PRL analyzed at specific times on day 6 are presented in Figure
20. In the cervically stimulated (CS) sham operated control rats
there were two peaks of prolactin release, one nocturnal (measured at

0300 hrs) and the other diurnal (measured at 1800 hrs). The nocturnal

94

.mo.ve u « .Honeem onu wo denon onu cmnu mmoH mH mm onn .naonm ono monHH on
ononB .mm H munomonaon ueHoe nooo nwsonnu oaHH HooHuno> onu new .monaom elm

wo woos onu muaomonmon neonw onu no uaHom noom .m eon no nouooHHoo moHaeom
wooHn neonu nH nonsmooe ono3 anooHone wo m:OHuonucoonoo eonom .cOHuoHsaHum
HooH>noo wnHSoHHow N eon no noenownoa moB A v enownsm eonm no A v eaouooHno>o

.omooHon aHuooHonm
wo anouuom onu no nOHuoHoeHum HmoH>noo wcH3oHHow eaouooHno>o wo noowwo one

.ON onomnn

95

 

 

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96
surge of CS ovariectomized rats was attenuated in length, but not
magnitude, and the diurnal surge was totally absent.

Adenohypophysial DA content of the same rats is presented in
Figure 21. Cervically stimulated sham operated controls showed peak
concentrations at midday (0900-1500 hrs) and again at 2400 hrs. A
significant nadir was measured at 0300 hrs and concentrations tended
to be decreased in the evening hours (1800-2100 hours).

There was also a cyclic variation in the adenohypophysial DA
content of CS-OVX rats. DA concentrations in the pituitary were low
in the early morning (0300-0600 hrs) and were elevated throughout the
remainder of the day in ovariectomized rats.

Significant differences in anterior pituitary DA concentration
between sham and OVX rats were detected at 0300, 1200 and 1800 hrs.
Throughout the evening hours (i.e., 1800-2400 hrs) OVX anterior pitui-
tary dopamine concentrations tended to be higher than sham concentra—
tions although only the 1800 hr difference was significant (p<.05).
These results suggest that pituitary DA content was decreased follow—
ing the surge release of prolactin.

The Effect of Ovariectomy Followinngervical Stimulation on DOPA
Accumulation in the Median Eminence

 

 

It has been suggested that PRL may cause a selective feedback
activation of TIDA neurons (Gudelsky 35 31., 1976; H5kfelt and Fuxe,
1972). Thus, the elimination of a surge of prolactin may be reflected
in decreased TIDA neuronal activity. To test this possibility, cervi-

cally stimulated rats were ovariectomized to eliminate the diurnal

 

97

.mHHouoo now oN onome ou onoon oom .muon nouoHseHum eHHoUH>noo ooNHaouooHno>o
wo enmuHsuHa noHnouno onu :H nonsmooa ono3 oGOHuonunoocoo oumumlenmoum oeHeomom

.muon
woumHseHum eHHooH>noolnoNHEouooHno>o CH mGOHuonucoonoo onHemaon enmuHsuHa nOHnouc<

.HN onownn

98

 

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99
surge of prolactin (Freeman e£_31,, 1974), and DOPA accumulation in
the median eminence was measured at selected times. On day 2 follow-
ing cervical stimulation, rats were randomly subdivided into 16 groups
of 8 rats each. Half of the groups were ovariectomized while the
remaining 8 groups received sham ovariectomy. A single blood sample
(approximately 1 ml) was collected from each rat by orbital sinus
puncture under light ether anesthesia at 0300 hrs day 5 post CS. The
following day, rats were sacrificed for determination of DOPA accumu- . f"

lation at one of the following times: 0300, 0600, 0900, 1200, 1800,

 

2100, or 2400 hrs. Rats in which the 0300 hr day 5 PRL concentration '5
was not greater than or equal to the mean minus 1 standard deviation
(E'- l S.D.) were excluded from the study. Table 10 lists the respec-
tive numbers of rats per treatment included in the analysis. Results
of the rate of DOPA accumulation in the median eminence are presented
in Figure 22.

The pattern of DOPA accumulation in the median eminence is simi-
lar for both sham and OVX rats except at 2100 hrs, when OVX values
were significantly decreased from control.

These results suggest that the absence of the diurnal surge of
prolactin at 1800 hr may result in decreased TIDA neuronal activity

due to a lack of prolactin feedback.

Discussion
An overwhelming amount of experimental evidence supports the con-
cept that the hypothalamus exerts a tonically negative control over
prolactin in release from the anterior pituitary. A major hypotha-

lamic inhibitory substance is DA, which is released from

100

TABLE 10

Number of Rats Per Treatment Cell for Determination
of DOPA Accumulation Following Cervical
Stimulation-Ovariectomy

 

 

 

TIME SHA‘MTreatment OVX
0300 3 8
0600 8 7
0900 7 7
1200 7 9
1500 7 5
1800 8 7
2100 7 7
2400 6 6

 

8Rats were cervically stimulated on the day of
estrus, and were ovariectomized during the
afternoon of the following day. A blood sample
was collected by orbital sinus puncture from
each rat at 0300 hrs day 5 following cervical
stimulation and rats were subjected to DOPA
accumulation determination at the listed times
on Day 6.

lOl

.mo.va u « .Honeem onu wo manon onu nonu mmoH mH mm onu .ezonm ono monHH

o: onon3 .mm H mueomonmon uaHom nooo nwaonnu onHH HooHuno> one .AOH oHnoe

oomv meoHuoeHanouon mum wo woos onu munomonmon neonw onu no uaHoa noom .A.e.H
.wn\we OOHV mHOH sz wo GOHuonumHnHaoo onu wcHsoHHow mHo>noucH nn N no noonHnomm
onoB moon .eoHuoHoeHum HooH>noo wnH3oHHow m eon co muon A v noNHaouooHno>o new

A V nouonoooiaonm wo ooeocHao noHnoa onu an noeHanouov mos GOHuoHnasooo <moa

.ooaoaHeo noHvoe onu an eOHumHsasooo
<moa wo anouuom onu no coHuoHseHum HooH>noo NGHBoHHow eEOUUoHno>o wo uoowwo one

.NN onowne

102

 

 

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TIME (hr)

 

103
tuberoinfundibular nerve terminals in the median eminence, directly
into the hypophyseal portal vessels for transport to the pituitary
(Ben-Jonathan, 1977). Consequently, alterations in tuberoinfundibular
dopaminergic neuronal activity may regulate the release of pituitary

prolactin. It has also been suggested that prolactin may in part

 

regulate its own release by activating the tuberoinfundibular nerves
through a sluggish feedback.mechanism (Gudelsky 33 31,, 1976). The
present experiment describes pituitary prolactin release during preg-

nancy and pseudopregnancy, and characterizes alterations in TIDA

 

neuronal activity when prolactin release is manipulated by restraint
stress or ovariectomy.

The results of the present study confirmed the biphasic release
of prolactin previously noted in early pregnancy (Butcher e£_31,,
1972) and pseudopregnancy (Freeman 3£_31,, 1974). Freeman e£_31,
(1974) also reported that the stress of surgery, blood collection, or
anesthesia resulted in the complete disappearance of the diurnal
prolactin surge of pseudopregnancy, although the nocturnal surge was
insensitive to such stressors. In contrast to those findings, results
of this study showed that both the nocturnal and diurnal prolactin
surges of pregnancy and pseudopregnancy are sensitive to stress.

Under our conditions, a 4 hr restraint stress imposed during the time
of the expected nocturnal surge of prolactin delayed the appearance of
the surge until later in the morning, when a surge of comparable

length and magnitude was detected. Furthermore, blood collection via
orbital sinus puncture did not diminish the appearance of the diurnal

surge of prolactin. The differences between the findings of the

104
present study and those of Freeman 33 31. (1974) may be due to the
more chronic nature of the stressors employed in the latter study.

A biphasic pattern of tuberoinfundibular dopaminergic neuronal
activity was also observed during pregnancy and pseudopregnancy.

Increased periods of neuronal activity occurred at times between the

nocturnal and diurnal surges of prolactin. The observed changes in

neuronal activity were specific for the tuberoinfundibular system

since no consistent changes were observed in other brain regions 7”
containing dopaminergic neurons examined. During a period when there
was no surge of prolactin (i.e., on the second day of diestrus), the
accumulation of DOPA in the median eminence did not change. This
finding suggests that there may be some relationship between the
surges of prolactin and changes in the TIDA neuronal activity Which
occur in pregnant and pseudopregnant rats.

Alterations in the surge release of prolactin are reflected in
changes of TIDA neuronal activity. When the administration of re-
straint stress delayed the nocturnal surge of prolactin in pregnant
rats, a shift in the peak activity of tuberoinfundibular dopaminergic
nerves was also observed. Furthermore, only one peak of TIDA neuronal
activity was detected in ovariectomized cervically stimulated rats in
which the diurnal prolactin surge was absent. These findings suggest
a causal role of the surge release of prolactin during pregnancy and
pseudopregnancy in the activation of TIDA neurons.

Attempts by other investigators to demonstrate a reciprocal rela-
tionship between hypophyseal portal blood DA concentrations and peri—

pheral prolactin levels have repeatedly failed (DeGreef and Neill,

105
1979; Ben-Jonathan ep 31., 1980). However, definite differences in
hypophyseal portal DA concentrations have been measured throughout the
day in cervically stimulated rats (DeGreef and Neill, 1979) and be-
tween stages of pregnancy (Ben-Jonathan, 1980). The limited number of
sample points, the time requirement to obtain a sample, and non—
specific anesthesia effects have all been suggested as possible reasons
for the failure to show reciprocity between portal DA and peripheral h
prolactin concentrations. However, decreased portal DA concentrations a

were measured during lactation when serum PRL concentrations are

 

elevated and the reverse was measured during late pregnancy (Ben-
Jonathan, 1980). Although these findings suggest that an inverse
relationship may exist between TIDA neuronal activity and prolactin
release, the feedback of placental luteotrophin on tuberoinfundibular
DA neurons is unknown. Recently, it has been suggested that rPL may
act at the hypothalamus to inhibit the release of prolactin (Yogev and
Terkel, 1978). Although the neuronal mechanism of this proposed
feedback is unknown, it may involve the activation of TIDA neurons.
Limitations in our experimental design do not allow for the correla-
tion of DOPA accumulation values to serum prolactin concentrations,
however, our results suggest that a "mirror image" relationship indeed
may not exist. It is possible that the TIDA nerve activity may follow
the surge release of PRL by a certain lag time dependent on other
factors.

Chiocchio 33 31. (1980) found that adenohypophyseal dopamine
content was inversely related to PRL release. In the present study,

steady-state concentrations of DA in the anterior pituitary of

106

cervically—stimulated rats also reflected prolactin release. In sham
operated controls, decreased concentrations of DA were measured at
times commensurate with the surge release of prolactin. Ovariecto-
mized animals, in which the diurnal surge was absent, showed only one
such nadir at 0300-0600, a time when the nocturnal surge was manifest.

The results of the present study support the hypothesis of an
Operational feedback loop between the surge release of prolactin which I»
follows cervical stimulation and TIDA neuronal activity. Although the w}
exact function of this relationship is not clear it is possible that

the prolactin induced neuronal activity may act to inhibit the release

 

of other hypophysiotropic hormones as well as supply the necessary DA
to inhibit the release of prolaction from the pituitary. Further
studies are required to determine if TIDA neurons have a causal role
in the initiation and maintenance of the daily prolactin surges

induced by cervical stimulation.

CHAPTER 4

Summary

Stress Effects on Pregnapey and Offspring Development

 

A primary hypothesis tested in these experiments was that mater-
nal stress would produce alterations in endocrine regulation of
reproduction leading to fetal wastage and possible alterations in
reproductive function of young rats exposed to maternal stress treat-
ment. The effect of maternal stress on fetal survival in this experi—
ment was much less than that of preliminary studies. In only one
treatment group was restraint stress sufficient to induce significant
incidence of abortion. In that situation, the females that did not
abort produced litters comparable in size to control females. Con-
sistent with this finding, Euker and Riegle (1973) reported that
restraint stress affected reproduction in an "all or none" fashion
such that females either aborted the pregnancy, or produced a litter
of normal size.

Results of several studies suggest that significant behavioral
disturbances develop in offspring of laboratory rodents which were
subjected to "psychological stress", e.g., conditioned response, while
pregnant (see review by Archer and Blackmun, 1971). Physical stress
e.g. immobilization or heat, of the pregnant rat has been associated
with a variety of measurable behavioral, anatomical and endocrinolo-

gical changes in male offspring (Ward, 1972; Dahlof 3£_31,, 1978a;

107

 

108
Politch ep_31,, 1978), however, few data are available concerning the
effects of prenatal physical stress on the developmental normalcy of
female rats. Results of the present study showed no abnormality in
development of prenatally stressed female rats. Prenatally stressed
females were found to develop normally in terms of weight, onset of
puberty, ovarian cyclicity, fertility and fecundity.

In contrast to these findings, it has been reported that prenatal
stress does result in decreased fertility and fecundity of female off-
spring (Herrenkohl, 1979; Herrenkohl and Gala, 1979). Such conflict-
ing reports are not unusual in the study of prenatally induced stress
effects. Archer and Blackmun (1971), addressed problems arising from
strain differences of rodents and treatment differences concerning the
type and duration of the stressor. They found that existing data were
difficult to interpret and concluded that few if any statements could
be made from the dozens of studies that they reviewed. Additionally,
since negative results tend not to be published, the existing data are
probably biased in favor of any debilitating effects of stress.

Another source of bias may be introduced into stress studies by
the use of littermates within the same treatment group. When using
siblings of polytocous species (e.g., rats, swine, dogs) in the same
treatment group, their similar genetic and environmental backgrounds
may result in a more uniform response to a treatment than if group
members were selected at random. It has been suggested that failure
to account for such a littermate effect could explain all of the

reported behavioral deficiencies of prenatally stressed male rats

 

 

109

(Chapman and Stern, 1978). The studies from Herrenkohl's laboratory
in which restraint stress was reported to reduce fertility and fecun-
dity of female offspring, do not address the allocation of pups to
treatment groups. It is doubtful that litter effects were considered
in her studies. Additionally, Herrenkohl's reports make no mention of
stress induced abortion. If the abortion rate due to stress was high,
one might assume that a greater chance exists for the use of surviving
littermates in subsequent studies. In the present study, the litter
effect was reduced by the minimization of littermates allocated to any

particular treatment group.

Prolactin Release and TIDA Neuronal Activity

 

The diurnal surge of prolactin during pregnancy is stress sensi—
tive (Riegle and Meites, 1976b), however, this study provided the
first demonstration that the nocturnal surge of pregnancy could be
altered by stress. Restraint stress administered during or before
either the nocturnal or diurnal prolactin surges of pregnancy can
block either PRL surge without affecting pregnancy. Results from a
time—course study following restraint stress administered from 2300
hrs day 5 of pregnancy until 0300 hrs day 6 showed that the nocturnal
surge of prolactin was not blocked, but rather delayed until later in
the morning.

Smith and Neill (1976a) suggested that the nocturnal prolactin
surge of pseudopregnancy was programmed to occur at a "critical
period", necessary to maintain the functional corpora lutea. Results

of the present study suggested that restraint stress reset the timing

 

110
of the surge. The resultant "nocturnal surge" was detected at 0900
hrs and appeared to be of normal magnitude and duration.

While little is known of the neuronal mechanisms that regulate
the surge release of PRL during pregnancy or pseudopregnancy, it is
generally accepted that dopamine released from tuberoinfundibular
neurons has a potent inhibitory effect on pituitary prolactin release
(MacLeod, 1976).

DA is released from the terminals of these nerves in the median
eminence where it enters the hypophyseal portal blood (Ben-Jonathan e;
31,, 1977) and is carried to the anterior pituitary. In the pitui-
tary, DA associates with prolactin granules (Nansel ep_31,, 1980). It
is thought that this DA then inhibits the release of prolactin from
the pituitary. In the present study, DA content of the anterior
pituitary reflected the surge release of prolactin in ovariectomized
and sham-ovariectomized cervically stimulated rats. These results are
consistent with the work of Chiocchio 3£_31, (1980) who demonstrated
an inverse relationship between serum prolactin levels and anterior
pituitary DA concentrations throughout the estrous cycle in the rat.

Results of the present study also indicated that there is a rela-
tionship between the surges of prolactin and activity changes of TIDA
neurons in pregnant and pseudopregnant rats. In control pregnant and
pseudopregnant rats, two surges of prolactin were measured, a noctur—
nal surge which peaked near 0300 hrs and a smaller diurnal surge
detected at 1800 hrs. TIDA nerve activity of pregnant and pseudopreg-
nant animals showed two peaks, one near midday, the other near mid-

night. When restraint stress during the expected nocturnal surge of

 

 

111
prolactin delayed the appearance of the surge by 3—6 hours, the apogee
of TIDA activity was likewise delayed. Ovariectomy of cervically
stimulated rats eliminated the diurnal surge of prolactin, and also
the late night peak of TIDA neuronal activity. Thus, the delay or
elimination of the surge release of prolactin, resulted in the delay
or absence of peak TIDA activity. These findings are consistent with
the current understanding of prolactin feedback on tuberoinfundibular

DA neurons (see Moore 33 31., 1980), and provide the basis for future

 

studies on the neuronal control of prolactin release during pregnancy.

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