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This is to certify that the

thesis entitled

THE EFFECT OF CONSTANT ILLUMINATION ON THE HYPOTHALAMO-
HYPOPHYSIAL-GONADAL AXIS OF THE FEMALE RAT

presented by

ROBE RT JOHN M I ODUSZEWSKI

has been accepted towards fulfillment
of the requirements for

Ph . D. degree in Physiology

 

 

 

Date July 21, 1978

 

0-7639

 

 

THE EFFECT OF CONSTANT ILLUMINATION ON THE HYPOTHALAMO-
HYPOPHYSIAL-GONADAL AXIS OF THE FEMALE RAT

By

Robert John Mioduszewski

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Physiology

1978

ABSTRACT

The Effect of Constant Illumination on the Hypothalamo-
Hypophysial-Gonadal Axis of the Female Rat

By

Robert John Mioduszewski

1. Mature Sprague-Dawley female rats were exposed to a 14:10 h
light:dark regimen (LB) or constant light (LL) for at least 5 weeks.

LL rats showing at least 10 consecutive days of vaginal estrus, or
regular 4-day cycling LD rats selected on proestrus were used in these
studies. Morning (0900-1000 h) and afternoon (1600—1700 h) serum

samples were obtained by decapitation from all animals. Basal serum
levels of follicle stimulating hormone (FSH) and prolactin (Prl), but

not luteinizing hormone (LH), were elevated in LL rats. In addition,

an afternoon serum FSH rise, but no preovulatory LH or Prl surge,

was seen in LL rats. These results suggest that constant illumination
may differentially block preovulatory LH and Prl surges while maintaining
a daily afternoon FSH release mechanism.

2. The pituitary response to single or multiple doses of syn-
thetic gonadotropin releasing hormone (GnRH) was compared in LL and
proestrous, estrous and diestrous LD rats in 3532, LL rat pituitary
LH and FSH release was not different from that of LD proestrous (PM)
rats, which showed the greatest pituitary sensitivity among those
groups of LD rats tested. It was concluded that constant light results

in a sensitization of the pituitary to GnRH.

Robert John Mioduszewski

3. Luteinizing hormone releasing hormone (LHRH) content was
measured in the anterior hypothalamic area (AHA) and medial basal hypo-
thalamus (MBH) of LL rats and LD proestrous rats in the morning and after-
noon. LHRH content was greater in LL than LD rats in both hypothalamic
areas and at both times. AHA LHRH content showed a decrease in the
afternoon of both LL and LD rats. However, whereas LD rat MBH LHRH
content decreased slightly in the afternoon, no change in MBH LHRH
content was seen in the afternoon in LL rats. These results suggest that
in the LL rat LHRH is synthesized and released from its site of synthesis
(AHA), but does not appear to be released from the MBH storage site.

4. Hypothalamic sensitivity to both positive and negative feed-
back by gonadal steroids was examined in ovariectomized LL or LD rats
primed with estradiol benzoate (EB) or progesterone (P). LL rat pitui-
tary LH, but not FSH, response to both negative and positive estrogen
feedback was less than in LB rats. In contrast, FSH, but not LH, re-
sponse to progesterone positive feedback was less in LL than LD rats.
These results suggest that LL rats may have a decreased sensitivity to
both positive and negative feedback by gonadal steroids on hypothalamic
control of gonadotropin release.

5. Plasma estradiol, progesterone and testosterone levels were
measured in LL and LD proestrous rats in the morning and afternoon.
Morning levels of serum estradiol were not different between LL and
LD rats; but unlike LD rats, LL rats showed no afternoon rise in plasma
estradiol. Plasma progesterone levels were higher in LD than LL rats
at both time periods. Plasma testosterone levels were low in the

morning as compared to the afternoon in LD rats, but the opposite pattern

Robert John Mioduszewski

in testosterone release was seen in LL rats. It was concluded that LL
rats have altered levels and patterns of plasma gonadal steroid release
which may contribute to an insufficient positive feedback effect on
hypothalamic gonadotropin‘ release.

6. Biogenic amine turnover index (T1) in LL rats was compared
with LD proestrous rats. Morning and afternoon biogenic amine TI was
measured in the preoptic-anterior hypothalamic area (AHA) and medial
basal hypothalamus (MBH) of all animals. Constant illumination resulted
in: a) a depression in norepinephrine (NE) TI and an increase in seroto-
nin (5-HT) in the AHA (AM), and b) a depression in the morning NE and
afternoon 5-HT TI in the MBH. It was concluded that constant light
may induce changes in hypothalamic biogenic amine metabolism associated

with a failure of LH release and increased Prl release.

ACKNOWLEDGEMENTS

I would like to express my gratitude to Dr. Joseph Meites, my
academic adviser, for his patience, direction and funding during the
course of my graduate work.

The counsel of my thesis committee members Drs. R. Dukelow, G.
Reigle, R. Pittman, R. Rech, and J. Ireland is also very much appre-
ciated.

Although their names are too numerous to list here, I am most
indebted to my friends and colleagues whose support played an invaluable

role in the progress of this work.

ii

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Page
ACKNOWLEDGEMENTS- — ii
LIST OF FIGURES vii
INTRODUCTION 1
LITERATURE REVIEW 5
I. Hypothalamic Control of Anterior Pituitary Secretion-- 5
A. Early Observations 5
B. Landmarks in Hypothalamic Anatomy 8
1. General Anatomy 8
2. Hypothalamic Nuclei and Their Afferent and
Efferent Connections 9
3. Hypothalamo-Hypophysial Blood Supply -------- 12
4. Transmission of Hypothalamic Impulses to
the Pituitary 15
C. Hypothalamic Hypophysiotropic Releasing and
Inhibiting Factors 16
II. Hypothalamic Monoamines 20
A. Pathways 20
B. Metabolism of Brain Amines 22
1. Biosynthesis 22
a. Catecholamines 22
b. S-Hydroxytryptamine 24
2. Storage . 24
3. Release 25
4. Catabolism 26
5. Uptake 27
III. Hypothalamic Control of Luteinizing Hormone (LH) and
Follicle Stimulating Hormone (FSH) Secretion----—----- 28
A. Neural Control Centers for Gonadotropin Release;
Cyclic vs. Tonic Centers 28
B. Monoamines as Transmitters of Gonadotropin
Release 32

iii

TABLE OF CONTENTS (continued)

IV.

MATERIALS

I.

II.

III.

IV.

 

l. Dopamine
2. Noradrenaline
3. Serotonin

 

 

C. Gonadal Steroid Feedback in Regulating Gonado-
tropin Release
1. Negative Feedback
2. Positive Feedback
3. Hypothalamic Sites of Feedback

 

 

 

 

The Effect of Light Upon Reproductive Function --------

 

A. General
1. Light as an Environmental Constituent-----—-
2. Characteristics of Natural Lighting ---------

B. Effects of Photoperiod on the Reproductive
Function

1. Pathways of Light Impulses Affecting Repro—
duction

2. Correlation with Reproductive Activity ------

3. Effect on Reproductive Cycle Length ---------

4. Effect on Sexual Maturation-

5 Effect on Timing of Ovulation and Mating
Behavior

6. Effect on Luteal Phase of Reproductive Cycle

 

 

 

 

C. Effects of Constant Light or Constant Dark on
Reproduction

 

 

AND METHODS

 

Animals, Treatments and Blood Collection

 

Radioimmunoassay of Serum Hormones

Assay of Hypothalamic Luteinizing Releasing Hormone
(LHRH)

 

A. Isolation and Preparation of Brain Tissue --------

B. Radioimmunoassay of LHRH

 

Assay of Dopamine (DA), Norepinephrine (NE), and
Serotonin (5-HT) in Brain Tissue

 

A. Isolation and Preparation of Brain Tissue --------

 

B. Radioenzymatic Assay of Catecholamines

 

C. Radioenzymatic Assay of Serotonin

iv

Page

32
37
39

47
47
48
50
52

52
52
52

53

54
56
57
57

57
59

6O
64
64

65

65
65
66

66
66
67
67

TABLE OF CONTENTS (continued)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Page
EXPERIMENTAL— 71
I. Effect of Constant Light on Morning and Afternoon
Serum Levels of Luteinizing Hormone (LH), Follicle
Stimulating Hormone (FSH), and Prolactin (Prl) in the
Mature Female Rat- 71
A. Objectives 71
B. Materials and Methods 71
C. Results 72
D. Discussion 74
II. Effect of Constant Light on the Sensitivity of the
Pituitary to Exogenous GnRH in vivo in the Mature
Female Rat; Single and Multiple Injections of Synthe-
tic GnRH— 76
A. Objectives 76
B. Materials and Methods 77
C. Results 78
D. Discussion— 84
III. Effect of Constant Light on the Content of LHRH in the
Anterior Hypothalamic Area (AHA) and Medial Basal
Hypothalamus (MBH) of the Mature Female Rat on Morning
and Afternoon Time Periods 86
A. Objectives 86
B. Materials and Methods 86
C. Results 87
D. Discussion- 89
IV. Effect of Constant Light on Hypothalamic Sensitivity
to Positive and Negative Feedback by Gonadal Steroids:
LH and FSH Response to Estradiol Benzoate (EB) and
Progesterone (P) Priming 91
A. Objectives 91
B. Materials and Methods 92
C. Results -- 93
D. Discussion 97

TABLE OF CONTENTS (continued)

V. Effect of Constant Light on Morning and Afternoon
Plasma Levels of Estradiol, Progesterone, and
Testosterone in the Mature Female Rat

A. Objectives—
B. Materials and Methods
C. Results- ‘

 

 

 

 

 

D. Discussion

VI. Effect of Constant Light on Hypothalamic Monoamine
Metabolism on Morning and Afternoon Time Periods:
Determination of Turnover Index of Norepinephrine (NE),
Dopamine (DA), and Serotonin (5-HT) in the AHA and MBH

A. Objectives-
B. Materials and Methods--—
C. Results-

 

 

 

 

D. Discussion-

GENERAL DISCUSSION-

 

 

 

 

 

APPENDICES
Appendix A. Coyle and Henry Catecholamine Assay Proce-
dure
Appendix B. Ben-Jonathan and Porter Catecholamine Assay
Procedure
Appendix C. Saavedra Serotonin Microassay

 

Appendix D. Steroid Radioimmunoassay

CURRICULUM VITAE

 

BIBLIOGRAPHY

 

vi

Page

99
99
100
100
101

106
106
107
108
113

118

126

126
128
130
132
134

137

Figure

10

11

12

LIST OF FIGURES

Degroot atlas section showing preoptic—anterior
hypothalamic area (AHA) and medial basal hypothalamus
(MBH) -

 

The effect of constant light on serum LH and FSH levels

in the morning (AM) and afternoon (PM)

 

The effect of constant light on serum Prl levels in
the morning (AM) and afternoon (PM)

 

The effect of constant light on pituitary LH response
to a single synthetic GnRH injection in_vivo

 

The effect of constant light on pituitary FSH response
to a single synthetic GnRH injection ig_vivo

 

The effect of constant light on pituitary LH response
to multiple synthetic GnRH injections ig_vivo ---------

The effect of constant light on pituitary FSH response
to multiple synthetic GnRH injections in_vivo ---------

Effect of constant light on LHRH (GnRH) content of the
preoptic-anterior hypothalamic area (AHA) and the
medial basal hypothalamus (MBH) in the morning (AM)
and afternoon (PM)-

 

The effect of constant light on estradiol benzoate
(EB) induced negative and positive feedback effects on
hypothalamic control of pituitary LH and FSH release--

The effect of constant light on progesterone (P) in-
duced positive feedback on hypothalamic control of
pituitary LH release

 

The effect of constant light on progesterone (P) in-
duced positive feedback on hypothalamic control of
pituitary FSH release

 

Effect of constant light on plasma estradiol and pro-
gesterone levels in the morning (AM) and afternoon
(PM)

 

vii

Page

69

72

73

79

80

81

82

88

94

95

96

102

LIST OF FIGURES (continued)

Figure

13

14

15

16

17

Effect of constant light on plasma testosterone in
the morning (AM) and afternoon (PM)

 

Effect of constant light on dopamine turnover index in
two areas of the hypothalamus in the morning and
afternoon -

 

Effect of constant light on norepinephrine turnover
index in two areas of the hypothalamus in the morning
and afternoon-----

 

Effect of constant light on serotonin turnover index
in the medial basal hypothalamus in the morning and
afternoon=

 

Effect of constant light on serotonin turnover index
in the preoptic-anterior hypothalamic area in the
morning and afternoon

 

viii

Page

103

109

110

111

112

INTRODUCTION

Light indicates time of day and time of year, and many species of
mammals appear to depend on its diurnal and annual variations for syn-
chronizing their reproductive activities (Hoffmann, 1973). The many
examples of continuation of reproductive function in the absence of
external cues suggest that endogenous rhythms are merely entrained to
one or more environmental signals, of which light seems to be the prin-
ciple one, but animals are not totally dependent on the external environ-
ment. The result is a reproductive cycle that provides the best chance
of survival for the species.

The system upon which light acts involves ultimately the gonads,
which produce the gametes and also the gonadal steroids. The gonads
are stimulated to grow and secrete steroid hormones by gonadotrophic
hormones released from the anterior pituitary gland. The pituitary
synthesizes and releases its gonadotrophic hormones under the influence
of neurohormones produced in the hypothalamus and released into the
hypothalamo-hypOphysial portal system in the region of the median emi-
nence. The neurohormones may stimulate or inhibit synthesis or release
of the gonadotropins from the pituitary. Although there is some evi-
dence for a direct effect of certain agents on the gonads, for the most
part the influences on the reproductive system act on the pituitary

itself or on the hypothalamic control centers.

2

The questions addressed by the experiments in this study relate to
the nature of changes that occur in several components of the hypothalamo~
hypophysial-gonadal axis in response to the absence of photoperiodic
change produced by constant illumination (LL) and resulting in persistent
estrus. Gonadotropin release is reported to be altered after constant
light exposure (Fink, 1975). There appear to be elevated levels of
serum follicle stimulating hormones (FSH) and prolactin (Prl), but no
pre-ovulatory luteinizing hormone (LH) Surge in light induced persistent
estrous rats. The latter may well be the major cause by which gonadal
function is altered. There may be other reasons for a change in the
"normal" pattern of gonadotropin release, not the least of which could
be an interruption of the neurochemical signals for gonadotropin release,
i.e., the gonadotropin releasing hormones (GnRH) of the hypothalamus.
There is a question of whether or not GnRH is synthesized and/or re-
leased from the hypothalamus into the portal blood system to stimulate
the pituitary in light induced persistent estrus. Therefore, hypothala-
mic GnRH content of LL rats was measured in these experiments. Another
possible cause for failure of ovulation could be reduced LH release due
to decreased sensitivity of the pituitary to GnRH. The proestrous stage
of the estrous cycle is characterized by an increase in pituitary sensi-
tivity to GnRH (Aiyer 35 al., 1973). If the pituitary of the LL rat is
not sensitive to the GnRH present, adequate LH release would not be
expected to occur. The pituitary sensitivity of LL rats to synthetic
GnRH was examined in 2339 in these experiments.

Since the gonadal steroids exert positive and negative feedback

effects on gonadotropin release (Everett, 1969; Brown—Grant, 1974),

3
constant light exposure may influence gonadotropin release indirectly
through changes in the amounts or ratios of serium estradiol, pro-
gesterone or perhaps even testosterone levels. Therefore, morning and
afternoon serum levels of these steroid hormones were measured in LL
rats.

The feedback effects which gonadal steroids have on gonadotropin
release is exerted both on the pituitary and the hypothalamus (Chowers
and McCann, 1967; Bogdanove, 1963). A change in sensitivity of the
hypothalamus to positive and negative feedback of gonadal steroids may
determine their effectiveness in inducing gonadotropin release or
inhibiting it at the proper time and in a cyclic pattern. Hypothalamic
sensitivity to estrogen and progesterone priming was examined in
ovariectomized rats under constant light (LL) or regular lightzdark
(LD) conditions.

Finally, there have been numerous reports implicating hypothalamic
monoaminergic neuronal control of gonadotropin release (Fuxe and
H6kfelt, 1969). The activity of hypothalamic monoaminergic neurons is
in turn influenced by steroidal feedback. Steroid feedback of gonado~
tropins may occur through mechanisms involving hypothalamic monoaminer-
gic neurons (Kalra gt 31., 1972). Constant light induced changes in
hypothalamic monoaminergic neuronal activity could eventually result
in a persistent estrous condition. Therefore, the hypothalamic turn-
over index (TI) of norepinephrine (NE), dopamine (DA) and serotonin
(5-HT) was determined in LL rats.

Functional changes in the above components of the hypothalamo-

hypophysial-gonadal axis were examined in light induced persistent

4
estrous rats, with the hope that this may contribute to a further
understanding of the mechanisms by which light influences reproductive

functions.

LITERATURE REVIEW

I. Hypothalamic Control of Anterior Pituitary Secretion

 

A. Early Observations

 

Early in the 20th century, clinicians recognized that pituitary
insufficiency might be related to damage in the region of the hypothala-
mus, but were unable to resolve whether the effects were those of direct
damage to the adjacent pituitary gland (Anderson and Haymaker, 1974;
Evans, 1935). On the basis of careful pathologic study, Erdheim (1909)
concluded that these changes could be caused by hypothalamic damage
alone, and Aschner (1912) demonstrated that gonadal insufficiency (now
recognized as being due to gonadotropin failure) could be produced in
dogs by hypothalamic lesions which spared the pituitary. Over the
following four decades, many workers studied the effects of "denervation"
of the pituitary by surgical section of the pituitary stalk, but the
results were ambiguous and controversial. In a series of classic experi-
ments, Harris and Jacobsohn (1952) demonstrated the crucial role of the
blood vessels of the stalk in this regulation. Stalk section in the rat
caused loss of sexual function, which returned when the hypophysial—
portal vessels regenerated. When a paper plate was inserted into the
stalk section so as to prevent regeneration of the vessels, sexual
function failed to return. In all animals, stalk section caused perma-
nent destruction of neural connection. They also repeated and extended

the earlier studies of Creep, who had shown that pituitaries transplanted

6
to the pituitary fossa function normally, whereas pituitary transplants
to other sites apparently were devoid of activity (Anderson and Hay-
maker, 1974). These observations showed that the pituitary fossa was a
privileged site for the growth and function of the pituitary, and indi—
cated that the crucial factor was the special blood supply from the hypo—
thalamus. Some criticized the results of the Harris-Jacobsohn experi-
ments as being due to damage to the pituitary caused by transplantation.
This reservation was resolved convincingly in the double transplantation
experiments of Nikitovitch-Winer and Everett (1958), who demonstrated in
rats that the pituitary failure resulting from tranSplantation of the
same pituitary to the region beneath the basal hypothalamus was not seen
if anatomic reconnection of the blood vessels occurred. Reconstitution
of pituitary function did not occur in control experiments in which the
pituitary was re-transplanted to the temporal lobe.

The local trophic function of the hypothalamus was also demonstrated
in the early 1960's by transplantation of pituitary fragments directly
into the hypothalamus. As reported independently by Halasz §£_al,
(1962) and by Knigge (1962), there is a region of the basal hypothalamus
that contains trophic substances capable of maintaining the cellular
structure and secretory function of the implants. The term "hypophy-
siotropic area" was applied to this region.

The hypophysial vessels themselves, now known to be the conduit
of the hypothalamic hypophysiotrophic hormones, were first described
in 1930 by a Roumanian medical student, Popa (1930), following the lead
given by his teacher, Ranier, in Bucharest. The vessels were charac-

terized by a peculiar group of coiled capillaries at the inferior extent

of the hypothalamus that left the brain and joined to form long vessels
that traversed the pituitary stalk. The blood in these vessels was in-
correctly asserted by POpa, and his subsequent coworker Fielding (pope
and Fielding, 1930), to flow from the pituitary upwards to the base of
the brain. In 1936, Wislocki and King described similar vessels in the
monkey and suggested on the basis of anatomic features that blood pro-
bably flowed from the hypothalamus to the pituitary. In 1947, Green
and Harris confirmed this suggestion by direct observation in the rat
that blood in the hypothalamic portal vessels did indeed flow to the
anterior pituitary, an observation which had been made independently
and previously in the toad by Houssay 33 31. (1935). Green and Harris
proposed that the hypothalamus secretes into the portal capillaries of
the median eminence specific pituitary-regulatory substances which are
transported to the adenohypophysis by the portal vessels. A similar
proposal was also made at about the same time by Friedgood (1936) in a
then unpublished lecture, and the earliest germs of this idea may be
found in the writings of Hinsey and Markee a decade before (Anderson
and Haymaker, 1974). However, more recent evidence by Hungarian (Palko-
vits, 1978) and American workers (Oliver g£_§1,, 1977; Bergland and Page,
1978) indicates that there is some retrograde flow of blood from the
pituitary to the hypothalamus. The significance of this retrograde
flow remains to be established.

Vesalius, in De Humani Corporis Fabrica (1543) described the
drainage of cerebrospinal fluid through the floor of the third ventricle
(named "infundibulum" because of its resemblance to a funnel) into the
pituitary and thence into the nose to form mucous (pituita) from which

our modern term pituitary is derived. More recent studies indicating

8
that certain hypophysiotrophic hormones may find their way to the an—
terior pituitary by way of the cerebrospinal fluid through the floor of
the third ventricle and median eminence indicate how leisurely, at
times, is the progress of scientific discovery.

Although the anterior pituitary gland lacks a direct nerve supply,
the secretion of each of its hormones is under the control of the central
nervous system. The pituitary and, in turn, its target glands respond
to changes in the external and internal environments through specialized
secretory neurons localized in the ventral hypothalamus. In addition,
the neurohumoral connections of the anterior pituitary are important in
the feedback regulation of a number of hormones, such as cortisol, the
gonadal steroids, and thyroxine, and serve as part of the integrated
mechanism by which behavior and metabolism adapt to the external environ-
ment.

B. Landmarks in Hypothalamic Anatomy

 

1. General Anatomy;

 

By definition, the hypothalamic region, though somewhat
ill defined, lies below the level of the thalamus. In effect, it
comprises the lateral walls of the lower part of the third ventricle,
below the level of the hypothalamic sulcus, which, in the adult human,
is a shallow and not very conspicuous groove running almost horizontally
along the lateral walls of the third ventricle. The rostral boundary
of the hypothalamus is indefinite, but may be taken as a plane lying
slightly rostral to the optic chiasm (the preoptic area). Its caudal
boundary may be taken as a coronal plane immediately posterior to the
mamallary bodies. The lateral boundaries of the hypothalamus are the

most difficult of all to define. At various coronal levels the

9
following structures may be found situated laterally; the very lowermost
part of the thalamus, the internal capsule, the globus pallidus, the
ansa lenticularis, and the optic tract. Below, the lowermost parts of
the gray matter making the walls of the third ventricle form, in man, a
funnel-shaped cavity, the infundibulum or tuber cinereum, a part of the
hypothalamus, which is clearly visible as a protuberance at the base of
the brain and which is prolonged downward as the pituitary stalk. This
region varies much in shape in other species of mammals, the part
corresponding to the anterior wall of the tuber cinereum and uppermost
part of the neurohypophysis having come to be called in most animals the
median eminence (Tilney, 1936). The pituitary gland is attached to
this region, either by means of a long, free-lying stalk, as in man,
primates, and the rat, or with virtually no stalk, as in the cat and dog.

2. (Hypothalamic Nuclei and Their Afferent and Efferent
Connections:

 

 

Within the confines of this rather vague region, the
hypothalamus, there are a number of very well-defined nuclear masses and
a number of remarkably ill-defined nuclear groups. There are also
several well-defined nerve fiber tracts and some diffuse tracts. One of
the most striking nuclei to be seen in the hypothalamus is the supra-
optic nucleus. This nucleus, which is extremely well defined and which
has a sharp outline, straddles the rostral extremity of the optic tract,
just caudal to the Optic chiasm. The axons of the nerve cells of the
supraoptic nucleus form a nerve tract composed of fine, unmyelinated
nerve fibers which pass through the rostral part of the tuber cinereum
or median eminence to run down in the neural part of the pituitary

stalk into the infundibular process or neural lobe of the pituitary

lO

gland. These axons form the supraoptico—hypophysial tract. It is
clear that a large number of these axons end in the infundibular pro-
cess, because when the pituitary stalk is cut many of the nerve cells
in the supraoptic nucleus degenerate, and disappear.

The paraventricular nucleus lies beneath the ependymal lining of
the third ventricle, dorsal and caudal to the supraOptic nucleus.

The axons of the cells of the paraventricular nucleus take a curved
course which passes toward the median eminence. This nerve tract com-
posed of fine unmyelinated nerve fibers is convex rostrally and passes
near or even through the supraoptic nucleus. The nerve tract formed by
the axons of these paraventricular nerve cells also forms part of the
hypothalamo-hypophysial tract and passes down through the median emi-
nence into the infundibular stem and the infundibular process (Daniel,
1966).

Ramon Cajal (1911) who first described the hypothalamo-hypophysial
tract, thought that it might have a sensory function. There was much
uncertainty about the tract until the work of the Scharrers (1954)
and Bargmann (1954) clarified its function. It was found that neuro-
secretory activity with specific staining properties was produced by
the cells of these two nuclei and that along the nerve fibers making
up the hypothalamo-hypophysial tract there could be found small bead
like swellings which also gave a specific staining reaction.

A prominent landmark at the base of the human brain, lying in
the interpeduncular space and immediately posterior to the tuber
cinereum is made by two prominent rounded swellings, the pair of
mammillary bodies. Two striking tracts of myelinated nerve fibers,

which are easy to see with the naked eye and can be easily dissected

11
out, are related to these nuclei. First, the fornix, a massive column
of myelinated nerve fibers, sweeps down obliquely through the hypo-
thalamic region from the superior rostral area to end in the mammillary
nucleus. This massive tract originates from the hippocampus and dentate
gyrus. The second massive and macroscopically visible nerve tract
associated with the mammillary nuclei is the mammillothalamic tract,
the main out-going tract which springs from the superior aspect of the
mammillary nucleus, passing upward and forward to enter and pass
through the lower parts of the thalamus. It ends in the anterior
nucleus of the thalamus. From the lowermost part of the mammillothalamic
tract a bundle of nerve fibers leaves the main tract to pass toward the
tegmental region of the midbrain as the mammillotegmental tract, which
joins the dorsal longitudinal fasciculus through which some of its
fibers are distributed through the reticular formation of the brain
stem.

The three nuclei so far described - supraoptic, paraventricular,
and mammillary, are easily identified histologically, being clearly
demarcated from the adjacent areas of the hypothalamus. The remaining
hypothalamic nuclei are far less well defined. In fact, some of them
only have been identified by using fetal material for preliminary
studies, since certain nuclei are well demarcated in the fetus although
become ill-defined in the adult.

The gray matter forming the walls of the third ventricle in the
region just above the tuber cinereum may be divided into two vertically
flattened plate-like nuclei, the ventromedial hypothalamic nucleus

and the dorsalmedial hypothalamic nucleus. These two nuclei lie in

12
the wall of the third ventricle between the attachment of the tuber
cinereum and the site of the paraventricular nucleus.

The ventromedial and dorsomedial nuclei do not seem to have any
particularly well-defined tracts of nerve fibers connected with them,
but there seems little doubt that they have connections with the other
hypothalamic nuclei by means of the extensive periventricular system
of nerve fibers and by means of the medial forebrain bundle, an ill-
defined sheet running anteroposteriorly along the length of the hypo-
thalamus and lateral to those hypothalamic nuclei which lie beneath
the ependyma of the third ventricle.

Above the mammillary nucleus and immediately caudal to the dorso-
medial and ventromedial hypothalamic nuclei, lies the posterior hypo-
thalamic nucleus also situated beneath the ependyma of the wall of the
third ventricle. The posterior hypothalamic nucleus appears to make a
considerable contribution to the periventricular system of fine nerve
fibers. These nerve fibers appear to form a pathway connecting many
of these hypothalamic nuclei with the ventricular aspect of the thalamus
above and with the brain stem below, the dorsal hypothalamic nucleus
perhaps providing the largest contribution. These seem to be some of
the main efferent pathways of the hypothalamus.

One more nucleus of ill-defined outline and composed of small
fibers may perhaps be mentioned. This is the preoptic nucleus lying
anterior to and above the level of the supraoptic nuclei.

3. _Hypothalamo-Hypophysial Blood Supply:

a. The Blood Supply of the Hypothalamus

 

The vascular supply for this region comes from

small arteries arising from the vessels of the circle of Willis (1e Gros

13
Clark, 1938) which, in effect, outlines the hypothalamic region on the
base of the brain. Some of the arterial twigs come from the superior
hypophysial arteries, some from the middle and anterior cerebral ar-
teries, and some from the posterior cerebral and communicating arteries.
b. The Blood Supply of the Pituitary Gland

The earliest useful work on the blood supply of the
human pituitary gland was that of Luschka in 1860, but it was not until
1930 that the subject of pituitary blood Supply became of special
interest. In this year, Popa and Fielding (1930) described a set of
vessels running along the pituitary stalk which were of a type not pre-
viously found in relation to the nervous system. These vessels formed
a connection between two capillary beds and thus had to be described
as portal vessels. Popa and Fielding (1930) considered that these
portal vessels carried blood from the pars distalis to the region of
the hypothalamus. These studies led to much work on the blood supply
of the pituitary, for it was at once obvious that a unique arrangement
of blood vessels such as they had described must be of considerable
functional importance. There was some evidence that the blood in the
portal vessels of the pituitary stalk did not flow from the gland
toward the brain, but in the opposite direction. Wislocki and King
(1936) studying the blood supply of the pituitary gland of the rhesus
monkey, came to the conclusion, on morphological grounds that the
direction of flow must be from the capillary bed at the top of the
stalk to the sinusoidal bed of the pars distalis. The direction in
which blood flowed along the portal vessels of the stalk was observed

in living animals, first by Houssay 55 El. (1935) and later by Green

14
23 31. (1947). However, as already mentioned, there is growing evidence
for some retrograde flow of blood from the pituitary to the hypothala-
mus.

Of all the advances in our knowledge of the pituitary gland over
the last thirty years, one of the most important has been that relating
to the control of its hormone secretion. The theory advanced by Harris
(1955, 1960) that the control of anterior lobe hormones is exercised,
at least in part, by means of neurohumors which are elaborated in the
hypothalamus and carried to the cells of the pars distalis by the blood
in the portal vessels, is now generally accepted. The importance of
this portal system throughout the vertebrate series was shown by Green
(1951) who studied a wide range of animals and found portal vessels
were present in all of them.

Two small arteries, the superior hypophysial arteries, spring
directly from the trunks of the internal carotid arteries in the sub-
arachnoid space, near to the origin of the opthalmic arteries. In
some cases these arteries arise not as a single small trunk, but as
twin arteries. In other cases the single trunk runs medially toward
the tuber cinereum or median eminence, dividing into two from an arterial
ring which encircles the upper extremity of the pituitary stalk. From
this arterial ring arises a series of branches, some of which supply
the optic chiasm and hypothalamic region. Others downward and inward,
penetrating the neural tissue of the median eminence and upper
infundibular stem, where they break up into a capillary bed of
complex patter. This had forms part of the first or primary capillary

bed of the portal system. The blood which has passed through these

15

coiled capillaries is collected into long portal vessels which pass
down the stalk. These superficial long portal vessels run through the
pars tuberalis. Other portal vessels pass down in the substance of
the neural tissue of the stalk. When the portal vessels enter the
pars distalis of the pituitary gland, they break up into the sinusoids
which form the blood supply of the epithelial cells of this lobe.

The neural tissue of the infundibular process or posterior lobe
of the pituitary gland is supplied by two arteries which spring from
the internal carotid arteries where these lie within the cavernous
sinuses, at the place where the carotid arteries turn sharply rostrally.
In the human, the artery of the trabecula passes through the epithelial
cells of the pars distalis to enter the lower infundibular stem, anasta-
mosing with a branch of the inferior hypophyseal artery and usually
taking part in some supply of the coiled capillaries which give rise
to the short portal vessels.

4. Transmission of the Hypothalamic Impulses to the
Pituitary:

When considering the connections between the hypothalamus
and the pituitary, the following well-established facts should be taken
into account: 1) There are no nerve endings of hypothalamic origin -
or at least occurring only exceptionally - in the pars distalis of the
hypophysis. An abundant hypothalamic innervation of the anterior lobe
tissue has been described by earlier authors, but a careful analysis
of their descriptions and illustrations proves undoubtedly that reti-
cular fibers have been mistaken for nerve elements. 2) The principal
blood supply of the anterior lobe is the portal system, which is charac—

terized by the intimate contact of the blood vessels with the nervous

16

tissue of the hypothalamus. 3) Hormone secretion of the anterior lobe
returns to normal after pituitary stalk section, if the vascular
connections between the hypothalamus and the pituitary have regenerated,
while pituitary and target organ atrophy develops, if for some reason
vascular regeneration does not occur (for details see Harris, 1955;
Szentégothaiugtial., 1968).

Based on the above-mentioned findings, Harris and Green (1947)
formulated the neurovascular hypothesis of adeno—hypophysial regulation.
They assumed that the hypothalamus produced some substances which,
after being released in the median eminence, enter the portal circula—
tion by which they are carried to the pituitary.

C. _Hypothalamic-Hypophysiotrophic Releasing and Inhibiting Factors

 

It has been shown by several authors that hypothalamic extracts
have a direct effect on pituitary function. Such an action was first
demonstrated on ACTH secretion (Saffran and Schally, 1955; Guillemin
and Rosenberg, 1955). The active principle was named corticotrOpic
releasing factor (CRF). Further studies suggested that hypothalamic
extracts contain substances which also influence the secretion of
other pituitary hormones. It was reported that hypothalamic extracts
stimulate the release of pituitary thyroid-stimulating hormone (TSH)
(Shibusawa gtflal., 1956a,b; Schreiber gt a1., 1961; Guillemin st 31.,
1962), luteinizing hormone (LH) (McCann gtflal., 1960; Campbell g£_al,,
1961; Nikitovitch-Winer, 1962), and follicle—stimulating hormone (FSH)
(Igarishi and McCann, 1964; Mittler and Meites, 1964). These substances
have been called TSH—releasing factor (TRF), LH releasing factor (LRF),

and FSH releasing factor (FRF), respectively. There is also evidence

17

that the hypothalamus produces a substance which exerts a tonic inhibi-
tory effect on prolactin release (prolactin-inhibiting factor, PIF)
(Pasteels, l96la,b; Meites SE 21., 1962; Talwalker g£_al,, 1963), a
growth hormone release inhibiting factor (GIF) (Krulich and McCann,
1968). The existence of these substances has been corroborated in
various laboratories by in 2222.33 well as in_vi££g_studies (see McCann
and Dhariwal, 1966; Schally gt 31., 1968a; McCann and Porter, 1969).

The functional significance of these factors is underlined by
the fact that concentration of the substances in the hypothalamus varies
in connection with changes in pituitary function. It has been shown
that during the sexual cycle of female rats there is a fluctuation
in hypothalamic LRF content (Ramirez and Sawyer, 1965; Chowers and
McCann, 1965). Suckling or estradiol treatment, besides influencing
prolactin secretion, cause a decrease in PIF activity of the hypothala-
mus (Ratner and Meites, 1964). Hypothalamic CRF activity shows a
diurnal rhythm similar to pituitary ACTH secretion. The daily peak of
CRF concentration in the hypothalamus is 3 hours earlier than the
daily corticosterone peak in the blood (David-Nelson and Brodish,
1969; Hiroshige g£_§13, 1969). Thyroidectomy was reported to produce
an increase in hypothalamic TRF and pituitary TSH content (Sinha and
Meites, 1965-66). There is more GRF in the hypothalamus of young
(30-day-old) than in old (2-year-old) rats (Pecile gt 31., 1965).

It appears that the hypothalamic hypophysiotropic substances are
responsible for both the structural as well as functional maintenance
of the cells of the anterior lobe. By tranSplanting it under the

kidney capsule or into the anterior ocular chamber, its structure

18
becomes dedifferentiated and composed mainly of chromophobic cells,
while its hormone content is markedly reduced (Cutuly, 1941; Cheng 33
'31., 1949; McDermott 25 31., 1950; Fortier, 1951; Harris and Jacobsohn,
1952; Siperstein and Greer, 1956; Goldberg and Knobil, 1957; Nikito—
vitch-Winer and Everett, 1959). Normal histology as well as function
of the dedifferentiated pituitary graft under the renal capsule can be
restored by retransplantation under the median eminence Nikitovitch-
Winer, 1958). These findings are consistent with the view that the
hypothetical hypothalamic substances exert a trophic influence on the
pituitary cells. It seems very likely that the action of the hypo-
thalamic substances are not only limited to release of the trophic hor-
mones, but that they also stimulate hormone synthesis. This is indicated
by the significant increase of pituitary TSH (Sinha and Meites, 1966;
Mittler gt 31., 1969) ACTH (Uemura, 1968), FSH (Justiz and de la Llosa,
1967), LH (Justiz £5 21,, 1967) and GH content (Symchowicz gt $1.,
1966; Schally gt_al,, 1968b) when under in 31532 conditions, hypothala-
mic extract or the appropriate releasing factor is added to the medium.
Median eminence extract administered intracarotidly causes a two to
five-fold increase in pituitary ACTH content (Vernikos-Danellis,
1965).

The chemical structure of three of the hypothalamic hormones has
been identified to date (TRF or TRH, LRF or LRH and GIF or somato-
statin). This has stimulated much investigation regarding their
localization and physiological action. TRH is found throughout the
hypothalamus and is not limited to any particular region. The

isolation and finally the chemical synthesis in 1969 of TRH (pyro

19

Glu-His-Pro-NHZ) (Schally 32 21., 1973; Guillemin £5 31., 1971) made it
possible to develop radioimmunoassays for this compound and methods
for detecting its biosynthesis. The highest hypothalamic concentration
of TRH was found in the median eminence. However, it is believed that
as much as 80% of TRH is found outside of the hypothalamus (Jackson
and Reichlin, 1974) mostly in cortical areas.

The decapeptide LRH (PYro-Glu-His—Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-
NH2) (Schally, 1971) is concentrated in the median eminence, extending
from the preoptic area (Alpert £5 31., 1976) and also the premammillary
area. It was found by immunocytochemical techniques that cells rostral
to the anterior commissure and paraolfactory area stain with anti-LRH
antibody. The lamina terminalis also contains LRH (Alpert g£_a1., 1976).

The third hypothalamic hormone to be chemically identific is soma-
tostatin (SRIF or GIF), a tetradecapeptide. The formula of this compound
is H-Ala-Gly-Cys-Lys-Asn-Phe—Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH with a
bridge between the two cysteine groups (Guillemin §£_al,, 1973). In
the relatively short time since its discovery, somatostatin has been
found to possess a surprising range of biological effects, to have an
unexpectedly wide anatomical distribution, and to be effective in all
species of mammals in which it has been tested. Somatostatin has been
shown to decrease TSH (Borgeat 25 a1., 1974), ACTH (Hall, 1973), GH
(Hansen, 1973), glucose and insulin (Koerker, 1974) and glucagon (Gerich
£5.2l3: 1975). Extrahypothalamic sites of somatostatin include the
pineal (Patel 33 31., 1975), pancreas, stomach, duodenum and jejunum

(Arimura gt 31., 1975).

20

Blood coming from the transected pituitary stalk has been demon-
strated to contain LRF (Fink 35 31,, 1966, 1967; Fink, 1967; Kamberi 35
31., 1969), FRF (Kamberi 25 31., 1970a), TRF, GRF (Wilber and Porter,
1970), CRF (Porter, 1970) and PIF (Kamberi 35 31., 1970b) activity.
According to Harris (1969), LRF is present in the portal blood during
all phases of the sexual cycle except estrus, and electrical stimulation
of the hypothalamus causes an increase in LRF activity of this blood.

It should be mentioned that in the peripheral blood of intact
animals the presence of these substances has not been conclusively demon-
strated, although earlier studies reported their presence in systemic
blood. This was only possible under special experimental circumstances
such as hypophysectomy, when presumably the releasing factors are re-
leased in larger amounts from the hypothalamus because of removal of the
inhibitory "short feedback 100p" from the AP hormones (CRF, Shapiro gt
.El" 1956; Eik-Nes and Brizzee, 1958; Brodish and Long, 1962; LRF,
Nallar and McCann, 1965; FRF, Negro-Vilar §£.E£" 1968a; GRF, Krulich
and McCann, 1966; Muller 33 31,, 1967; CRF, Redding and Schally, 1969;
PIF, Chen gt $1., 1970). If the medial basal hypothalamus is destroyed,
the releasing or inhibiting activity in the peripheral blood of the
hypophysectomized animals was not evident, suggesting a hypothalamic

origin of the active principles.

II. Hypothalamic Monoamines

 

A. Pathways

The regional distribution of catecholaminergic neurotrans-
mitters in the brain and spinal cord was determined imprecisely, using

biochemical assays, many years ago; however, the more recent application

21

of histochemical fluorescence methods has made it possible for these
compounds to be visualized within specific neuronal tracts and cell
bodies (Dahlstrom and Fuxe, 1964; Fuxe, 1965; Andén 33 31., 1966). With
the use of this approach, dOpamine appears to be concentrated in three
major groups of brain neurons: nigrostriatal neurons terminating in the
basal ganglia; meso-limbic neurons, with cell bodies localized in the
upper brain stem and axon terminals in the nucleus accumbens, olfactory
tubercles, and cerebral cortex; and tuberoinfundibular neurons, located
entirely within the hypothalamus and having their most prominent set
of terminals in the external layer of the median eminence (Fuxe and
Hokfelt, 1969; Jonsson 25.31., 1972). Nerve tracts that appear, by
histochemical fluorescence assay to contain norepinephrine as a neuro-
transmitter are also grouped into three main fiber bundles; a descending
system, with cell bodies localized in the medulla oblongata, whose
axons course through the spinal cord to form synapses with ventral and
dorsal horn neurons; and two ascending tracts, one with cell bodies
located in the locus coeruleus and terminals in the cerebral cortices
and the hippocampus, and a second tract with cell bodies in the medulla
and pons and axons terminating largely within the hypothalamus. Within
the hypothalamus, norepinephrine can be detected by microenzymatic
assay in each nucleus, with the highest concentrations in the median
eminence and arcuate nucleus (Palkovits g£_al,, 1974).
Serotonin-containing neurons are only poorly visualized with the
histochemical fluorescence method; however, available data seem ade-
quate to justify the conclusion that virtually all of the serotonin

cell bodies in brain are confined to the mid-line raphé nuclei of the

22

medulla and pons. Fibers from these cell bodies descend to terminate
in the spinal cord gray matter and ascend in the brain (mainly via the
medial forebrain bundle, along with dopaminergic neurons and noradre-
nergic axons) to innervate most of the telencephalon and diencephalon.
Most hypothalamic nuclei contain serotonin (and thus, probably 5-HT
terminals), as assayed by microenzymatic techniques (Saavedra §£_a1.,
1974). The highest concentrations are found in the arcuate and supra-
chiasmatic nuclei, with smaller, but nonetheless substantial amounts
are present in the median eminence.

B. Metabolism of the Brain Amines

 

l. Biosynthesis

 

a. Catecholamines:

 

The synthesis of catecholamines takes place within
neurons from tyrosine. The distribution of tyrosine hydroxylase which
converts tyrosine to dopa parallels the distribution of catecholamines
in the brain; its concentration is high in the hypothalamus, midbrain,
pans and striatum and low in the cortex (McGreer g£_al,, 1967). It is
found partially free and partially bound to membranes, the latter form
being more active (Costa and Meek, 1974). L-aromatic amino acid decar-
boxylase is found only in the soluble fraction of nerve tissue and is
present in all parts of the central nervous system (CNS). Dopamine-B-
hydroxylase which converts dopa to noradrenaline is associated with
the synaptic vessicles in which the amines are stored. It is found in
high concentrations in the midbrain and hypothalamus, but is low in the
striatum (Basal ganglia) where dopamine is the main transmitter

(Iversen and Glowinski, 1966).

23

The rate-limiting step for catecholamine synthesis depends on the
activity of tyrosine hydroxylase which is under a negative feedback
control exerted by "releasable" noradrenaline. When this is removed,
not only does the activity increase but there is also an increase in
synthesis of tyrosine hydroxylase (Spector 25 $1., 1967; Weiner and
Rabadjija, 1968). This negative feedback effect was first shown by
Costa and Neff (1966); they raised noradrenaline levels in the brain by
inhibition of monoamine oxidase and within 1 or 2 hours of this treat-
ment noted a fall in turnover rate. Similar effects have been shown for
dopamine (Javoy £5 31., 1972). Thus, it can be expected that the turn-
over rate will differ in the various areas of the brain, since the sizes
of the storage pools are different. For example, the turnover rate for
noradrenaline is slowest in the hypothalamus (t% = 4 hours), where its
storage and uptake is greatest (Iversen and Glowinski, 1966).

The pattern of control of catecholamine synthesis in the brain is
very similar to that in the adrenal medulla, where the first investi-
gations were made. The adrenals differ, however, in that they contain
high concentrations of phenylethanolamine-N-methyl-transferase (PNMT),
the enzyme which converts noradrenaline to adrenaline (Axelrod, 1962).

Although levels of adrenaline in the brain are negligible, the
enzyme PNMT is present especially in the hypothalamus and olfactory
bulb. In addition, an inhibitor of PNMT can be found in the same areas,
which may explain the low levels of adrenaline in the brain. The pre-
sence of the enzyme and its inhibitor suggest that adrenaline is syn-
thesized in the brain, it is locally regulated and is not stored

(Ciarnello gt $1,, 1969; Pohorecky g£_al., 1969).

24

b. 5—Hydroxytryptamine

 

The synthesis of 5-hydroxytryptamine from tryptophan
also takes place inside the neurones, under the control of tryptophan
hydroxylase and L-aromatic amino acid decarboxylase. The distribution
of tryptophan hydroxylase correlates with the distribution of neurones
liberating 5-hydroxytryptamine at their synapses (Ichiyama §£_al,,
1970). The hydroxylation was thought to be the rate-limiting step of
the synthesis (Moir and Eccleston, 1968), but recent evidence has shown
that the rate depends on the availability of tryptophan and its transport
across the neuronal membrane since normal brain levels of tryptophan
do not saturate the tryptophan hydroxylase (Grahame-Smith, 1971; Taglio-
monte gt 31., 1971).

Whether the level of 5-hydroxytryptamine at nerve terminals can
exert a negative feedback effect on its own rate of synthesis is yet
to be elucidated. Administration of a monoamine oxidase inhibitor re-
duces the conversion of 3H—S-hydroxytryptophan to 3H—S-hydroxytryptamine
(Macon E£.§l:: 1971); however, this result has been questioned (Costa
and Meek, 1974) since the change in specific activity with time was not
taken into consideration. Indeed, Costa and Meek (1974) have shown that
there is no change in turnover rate after increasing 5-hydroxytryptamine
brain levels by monoamine oxidase inhibition.

2. Storage
The storage of amines in the brain is similar to that in

peripheral nerves; they are stored either in a "bound" form or in an
"easily-releasable", that is to say "functional" store (Aprison and

Hingtgen, 1972). As the most newly synthesized transmitter is the most

25
easily released (Hamon £5 31., 1970; Farnebo §t_al,, 1971), it seems
that the amines must fill the "functional" store before filling the
"bound" store. Electron microscope studies have shown that the amines
are present in small granulated vessicles in "boutons" (swellings) of
the nerve terminals, and these are presumably the storage sites
(Kobayashi and Matsui, 1969), although the anatomical distribution of the
bound and functional stores are not known. The storage granules are
thought to be formed in the cell bodies of the neurones and to pass down
the axon to the nerve terminals. This has been shown by noting changes
in histofluorescence after ligaturing noradrenergic axons (Dahlstrom,
1967).
3. Release
Each amine is released from its nerve terminal after an

action potential. This has been shown to occur igflyitgg after stimula-
tion of cortical slices (Baldessarini and Kopin, 1967). The release of
both noradrenaline and S-hydroxytryptamine from the brain is dependent
on calcium ions (Goodwin g£_§1,, 1969). Release of the amines after
electrical stimulation has also been shown in_!izg, Stimulation of
the ventral noradrenergic bundle in the midbrain causes a fall in nor-
adrenaline in the hypothalamus (Arbuthnott 35 al., 1970) and stimulation
of the midbrain raphé causes a fall in 5-hydroxytryptamine in the fore-
brain with a concomitant rise in its degradation product, 5-hydroxy-
indole acetic acid (5-HIAA) (Aghajanian g5_§l,, 1967; Sheard and
Aghajanian, 1968).

Electron microscopy shows that the amines are released from their
storage sites by a process of exocytosis in which the contents are

released into the extraneuronal space; for example, at noradrenergic

26
nerve terminals, noradrenaline, dopamine—B-hydroxylase and Specific
proteins have been shown to be discharged after stimulation (Malamed gt
‘al., 1968).

The free or active transmitter, after its release, acts on the

receptor sites of the post-synaptic neuron or organ.
4. Catabolism

Noradrenaline and depamine are removed from their sites
of action mainly by an efficient uptake system back into the nerve ter-
minals, where they are either restored or metabolized by the intra-
neuronal enzyme, monoamine oxidase, which is found associated with the
mitochondria (Schnaitman gt_al,, 1967). Monoamine oxidase has no re-
gional Specificity but has its highest concentrations in the hypothalamus
(La Motte 35 31., 1969). Approximately 10 percent of the catecholamines
are metabolized extraneuronally by catechol-O-methyltransferase (COMT)
(Axelrod, 1959) which converts noradrenaline to normetanephrine and
dopamine to 3-methoxytyramine.

The system is similar for 5-hydroxytryptamine in that there is a
specific reuptake system, so that 5-hydroxytryptamine is either restored
or metabolized by monoamine oxidase to 5-HIAA. There are differences
as COMT does not degrade tryptophan derivatives, but enzymes are found
almost exclusively in the pineal gland for converting 5-HT to melatonin.

Mere recently, two other enzymes that metabolize 5-HT have been
found in the brain. One is N-methyltransferase enzyme (Mandell and
Morgan, 1971) which has been identified particularly in the pituitary
and the pineal and overts 5—hydroxytryptamine to its methyl derivatives,
which may be psychogenic (Himwich, 1971). The other enzyme is 5-hydroxy-

tryptamine sulphotransferase which has been found in the soluble

27

fraction of neuronal tissue and which convert 5-HT to 5-HT-O-su1phate
(Hidaka £5 31., 1969).

5. Uptake

Uptake mechanisms for inactivation of noradrenaline were

first shown in peripheral nerve endings (Iversen, 1965) but have since
been shown to occur in the brain for noradrenaline, dapamine and 5-
hydroxytryptamine. Uptake in zi!g_was demonstrated either by accumula-
tion of radioactivity labeled amines at specific sites in the brain
after intraventricular injection (Glowinski g£_a1,, 1965; Glowinski and
Iversen, 1966) or by noting the increase in fluorescence in specific
sites after intraperitoneal injection of the amines (Lichtensteiger and
Langemann, 1966; Fuxe 25.31., 1968b). These sites were usually those
which normally contained high endogenous levels of the particular trans-
mitter. In the case of noradrenaline 80 percent of the uptake of exo-
genous amines occurs into brain nerve terminals, the rest is accumulated
by a low affinity mechanism into other brain tissue (Baldessarini and
Vogt, 1971).

Dopamine can be taken up by noradrenergic neurons in the central
nervous system and either converted to noradrenaline within 15 to 30
minutes (Glowinski and Axelrod, 1966) or possibly act as a false trans-
mitter (Farnebo st 21., 1971). Dopamine also has a specific uptake
system of its own. The uptake of 5-HT in the brain has been demon-
strated in gizg and in 23533 (Schanberg, 1963). The uptake occurs at
two sites; in low concentrations it enters specifically into tryptami-
nergic nerve endings, but in higher concentrations it accumulates into
catecholaminergic neurons and catecholamines and 5-HT compete for the

same uptake sites (Shaskan and Snyder, 1970).

28

III._Hypothalamic Control of Luteinizing Hormone (LH) and Follicle
Stimulating Hormone (FSH) Secretion

 

 

A. Neural Control Centers for Gonadotropin Release; "Cyclic"
vs. "Tonic" Centers

 

 

There is considerable evidence that a structure adjacent to

the hypothalamus is of importance for the normal cycling activity in
female rats. This area, the parvicellular medial preoptic area (MPO),
receives rich input from limbic and mesencephalic nuclei (limbic-midbrain
circuit) (Nauta, 1958; Szentagothai g£.al., 1962; Heimer and Nauta, 1969;
Nauta and Haymaker, 1969). The medial preoptic area of female rats
binds labeled estradiol (Stumpf, 1971; Pfaff and Keiner, 1973) and shows
in electron micrographs distinct differences in the distribution of
different types of synapses when compared to the MP0 of the male rats
(Raisman and Field, 1971). Electrolytic lesion of the MPO stops estrous
cycle activity (Everett and Radford, 1961) and electrochemical or elec-
trical stimulation results in increased pituitary gonadotropin and pro—
lactin release (Wuttke g£_al,, 1972; Kalra gt 31., 1973). If the fibers
connecting the MPO and the medial basal hypothalamus (MBH) are dis-
connected by the use of a small bayonet-shaped knife (Halasz-knife),
normal cyclical activity also disappears (Taleisnik.g£flal., 1970; Koba-
yashi and Miyake, 1971). Whereas a more rostral cut, which disconnects
the fiber inputs to the MPO but leaves the fibers between the MPO and
MBH intact, has no severe effects on the estrous cycle (Koves and Halasz,
1970).

For all these reasons it seems reasonable to consider the MPO as
an important structure regulating the cyclical release of LH and possibly
prolactin. Thus, the MPO is usually referred to as the "cyclical" center

as opposed to the "tonic" center, which is located in the MBH and

29
regulates tonic pituitary LH release (Gorski and Wagner, 1965). Both
areas, the MPO and the MBH are sites of feedback interactions (Stumpf,
1970). Lesions in the MBH (tonic center) result in anovulation asso-
ciated with permanent anestrus indicating reduced pituitary gonadotropin
release (Igarashi and McCann, 1964; Bishop £5 31., 1972). Lesions of
the MPO (cyclic center) also stop cyclical activity but lead to a con—
stant estrus syndrome with polyfollicular ovaries but no corpora lutea
(D'Angelo and Kravatz, 1960; Flerko and Bardos, 1960).

The MBH is the part of the diencephalon where the releasing and
inhibiting factor (RF and IF) producing neurons are considered to be
located. With the help of two different methods, Halasz and associates
gave evidence that the hypothalamic hormones are produced in the so-
called hypophysiotropic area of the hypothalamus. Stereotaxic implanta-
tion of pituitary fragments into various hypothalamic structures of
hypophysectomized and castrated rats results only in the occurrence of
castration cells in the fragments if hypothalamic hormones exert their
action on these fragments. Thus, regions which contain hypothalamic
hormones could be mapped. It was demonstrated that only the mediobasal
part of the hypothalamus has hypophysiotropic properties (Halasz gt
.al., 1962; Knigge, 1962). Later, using his knife technique, Halasz
and Pupp (1965) and Halasz and Gorski (1967) isolated the mediobasal
hypothalamus from the rest of the brain. This complete deafferentation
resulted in disruption of estrous cycle activity because the medial
preoptic area had been disconnected from the mediobasal hypothalamus.
Basal pituitary hormone secretion was unchanged though, indicating that

the basal secretion of the hypothalamic hormones was still intact.

30

Whether the LH release controlling neurons are only present in the
mediobasal hypothalamus or whether these neurons are also present in
the preoptic areas as suggested by Wheaton gt 21. (1975) in the rat and
by Barry g£_al, (1974) in the guinea pig remains questionable. The
presence of LRH containing neurons in the preoptic area, however, does
not necessarily mean that it is these neurons that are involved in pre-
ovulatory gonadotropin secretion because the synthetic decapeptide LHRH
has been shown to have effects on mating behavior (Moss and McCann,
1973, 1975).

The very basal part of the MBH is the arcuate nucleus where clusters
of dopaminergic neurons are located with fiber projections into the median
eminence (ME) (Carlsson gt 31., 1962; Fuxe, 1963). Other axon terminals
from noradrenergic and possibly serotonergic fibers arriving from mesen—
cephalic structures are also found in the ME (Fuxe, 1965; Andén g£_al,,
1966; Ungerstedt, 1971). These terminals and most likely axonal endings
from hypothalamic factor-producing neurons form intimate contact with
the portal vessels that run along the ME and the pituitary stalk linking
the hypothalamus and the anterior pituitary gland.

It has been shown that spontaneous activation of the "cyclical"
center occurs a relatively short time before the preovulatory LH surge
is released. This spontaneous activation can be inhibitied by drugs
such as anesthetics (e.g., pentobarbital), and also drugs that modify
the metabolism of certain monoamines (Everett g£_al,, 1949; Everett and
Sawyer, 1950). If pentobarbital anesthesia is initiated prior to the
activation of the cyclic center, this activation will be inhibited, the
preovulatory LH and Prl surge is abolished and ovulation will not occur

(Wuttke and Meites, 1970). The most effective period for pentobarbital

31

inhibition 0 f the cyclic center is the early afternoon, a few hours
prior to the time when the LH surge normally occurs. The most effective
period has been called the "critical period". These pentobarbital-
blocked proestrous rats provide a good model to study the effects of
electrical stimulation of the MPO or hypothalamic structures (Bunn and
Everett, 1957). Critchlow (1958) was the first to do this type of
experiment. Electrical stimulation of the MBH proved to be effective in
restoring ovulation in pentobarbital-blocked rats. Later Everett and
Radford (1961) demonstrated the same effect after stimulation of the MPO.
It is now well established that electrical or electrochemical stimula-
tion of the female MPO results invariably in pituitary gonadotropin re-
lease, which is probably due to increased release of hypothalamic LRH
into the portal vessels (Harris and Ruf, 1970). The magnitude of
response, however, varies with the stage of the estrous cycle (Kalra
and McCann, 1973). Highest post-stimulatory LH levels were found in
proestrous rats, which again demonstrates the priming effect of
estrogens (Kalra and Kalra, 1974). It was also shown that injection of
progesterone into rats on the mroning of proestrus advanced the
critical period and the time of preovulatory gonadotropin release
(Zeilmaker, 1966; Nallar gt 31., 1966; Caligaris g£_§1,, 1968; Lawton,
1972). These observations certainly suggest that differences in
neuronal activity in the MPO should be demonstrable at different re-
productive stages. A number of attempts have been made to record such

neuronal activities under different endocrine conditions.

32

B. Monoamines as Transmitters of Gonadotropin Release

 

1. Dapamine

The results obtained after altering levels of hypothalamic
dopamine fall into two categories. The earlier work of McCann and his
coworkers suggested that this amine stimulates gonadotropin release,
while there is now accumulating evidence Suggesting it inhibits gonado-
tropin release.

In 1969 McCann and his coworkers started an extensive investigation
into the effects of amines on gonadotropin release. They incubated
rat pituitaries with each of the three amines in turn and found that
dopamine had no direct effect on the release of gonadotropins but that
adrenaline enhanced the release of FSH and LH, and noradrenaline only
enhanced FSH release (Schneider and McCann, 1969a; Kamberi and McCann,
1969a,b; Van Loon and Kragt, 1970; Quijada 25.31., 1974). When portions
of the stalk-median eminence area of the hypothalamus were added to
the incubation, the release of both FSH and LH was enhanced by dopamine
even at very low concentrations (0.5-5 ug/ml) while noradrenaline and
adrenaline had no effect (Schneider and McCann, 1969b; Kamberi 35 31.,
1970a). Dopamine must have acted in these experiments by stimulating
the discharge of LH and FSH-releasing hormone from the hypothalamic
fragments since it did not potentiate the action of exogenous LH/FSH
releasing hormone added to the incubation mixture. aeAdrenergic and
dopaminergic blocking agents (phenoxybenzamine, phentolamine and halo-
peridol) inhibited the release, and a B-adrenergic blocking agent (pro-
pranolol) did not (McCann ggnal., 1972). The prior addition of estradiol
to the incubation also blocked the releasing effect of dopamine and

Schneider and McCann (1970a) suggested that estradiol exerts its negative

33
feedback effect in physiological conditions by inhibiting the stimulating
action of dopamine on LH/FSH-RH release.

The stimulating effect of dopamine has also been shown in ig_!igg
experiments. Intraventricular injection of 1-4 ug of dOpamine increased
the LH/FSH releasing hormone activity in the plasma taken from the hypo-
physial portal system (Kamberi ggflal., 1969a, l970b,c, 1971a) as well as
the peripheral plasma of hypophysectomized rats (Schneider and McCann,
1970c), and this stimulatory effect was prevented by pretreatment with
estradiol (Schneider and McCann, 1970c). Similarly, dopamine stimulated
the release of LH and FSH into the circulating blood of intact male and
female rats and in ovariectomized female rats pretreated with ovarian
hormones (Schneider and McCann, 1970d; Kamberi 35 51., l970a; Porter gt
.31., 1972). In the intact female rat, dopamine was particularly effec-
tive on the second day of diestrus and on proestrus, and these effects
were reversed by a-adrenergic blocking agents (Schneider and McCann,
1970d). Parallel experiments using adrenaline and noradrenaline re—
vealed that both amines given intraventricularly stimulated gonadotropin
release but that slightly higher doses (2.5-5 ug) were required and
that doses of 100 ug or more were required for the two amines to be
effective in intact males (Schneider and McCann, 1970b,c,d; Kamberi 35
31., 1970b).

When the effective intraventricular dose of dopamine was injected
directly into the portal hypophysial system, release of gonadotropins
was not Stimulated, thus demonstrating as in the in_zi££g_experiments
that dopamine did not act directly on the pituitary (Kamberi g£.a1.,

1970b). Dopamine was also inactive when perfused into the arteries

34
supplying the anterior and posterior areas of the median eminence,
indicating that the median eminence is probably not the site of action
either (Porter 25 31., 1972).

Other experiments have confirmed that depamine is of major impor-
tance for the release of gonadotropins. Kordon and Glowinski (1969,
1970) injected a-methyltyrosine and a-methyldopa into immature rats
induced to superovulate by pretreatment with PMS and HCG. They found
that these inhibitors of catecholamine synthesis blocked ovulation when
given during the critical period but not if given a few hours before.
This is strange as these compounds have an onset of action of 2-3 hours
and a duration of up to 24 hours. DOpa (which restores the levels of
both dopamine and noradrenaline) partially reversed the effect of a-
methyltyrosine, and dihydroxyphenylserine (DOPS) (which restores nor-
adrenaline levels only) had no effect on the ovulation. These experi—
ments suggest that a-methyltyrosine inhibited ovulation due to a reduc-
tion of dopamine synthesis during the critical period. Kordon (1971)
also found that an intrahypothalamic implantation of a-methyltyrosine
inhibited ovulation, but only when placed in the arcuate nucleus-median
eminence region, the site where, in fact, the dopamine neurons termi-
nate. In a separate study pimozide (a DA receptor blocker) was shown
to inhibit the release of LRH in hypophysectomized rats (Corbin and
Upton, 1973).

A directly opposite role for dopamine has been suggested by Fuxe
and his coworkers. They showed that there was an increased dopamine
activity in the median eminence at the times when gonadotropin release

was inhibited. It is possible, as has been suggested by Fuxe and

35

Hfikfelt (1970) and even by McCann (Donoso SE 31., 1971), that dopamine
is converted into noradrenaline after intraventricular injection and
only appears to be more active than noradrenaline because injected nor-
adrenaline is rapidly metabolized. This idea is supported by the fact
that phenoxybenzamine, which blocks a-adrenergic and not dopaminergic
receptors (Fuxe and Hfikfelt, 1970), antagonized the gonadotropin releasing
action of dopamine. Also, dopamine infusions are effective in releasing
gonadotropins only when infused into the median eminence where dopami-
nergic nerve terminals are situated (Porter 22 31., 1972). Porter 25 31.
(1972) has found that intraventricular injection of saline can induce
gonadotrOpin release perhaps due to mechanical stretching of the ven-
tricles. It is possible that the results obtained with dopamine in 3119
are due to this artifact.

The results Supporting the hypothesis that dopamine stimulates the
release of the gonadotropins have been widely published, but in fact
all the experiments in which dopamine itself was used, either ig_zigg
or in gigrg, came from McCann's laboratory between 1969 and 1971. In
1974 they repeated their in zi££g_experiments by adding dopamine to
incubating pituitaries and hypothalamic fragments and found no effect
on gonadotropin release; the only difference between this experiment
and the previous ones was that three hypothalamic fragments were used
instead of two (Quijada 35 31., 1974). In a Similar type of experiment
in which pituitaries attached by an intact portal system to a median
eminence were superfused, the presence of 2.5 ug/ml of dopamine inhi-
bited gonadotropin release (Miyachi 23 31., 1973).

Some ig_yigg_experiments have also shown that dopamine can inhibit

gonadotropin release. In an acute experiment, 80 ug of dopamine was

36

infused into the arcuate nucleus during the critical period in cyclic
rats and found to inhibit ovulation (Craven and McDonald, 1973). In
another report, implants of dopamine were placed into the median emi—
nence area (as well as other parts of the hypothalamic) and were shown

to have an inhibitory effect. Uemura and Kobayashi (1971), found that
implants of dopamine in cholesterol (2:1) left for two weeks in the
posterior part of the median eminence (which included part of the arcuate
nucleus) appeared to suppress the cyclic release of LH, and both the
cyclic and tonic release when the proportion of 5 to 1. These two im-
plants were estimated to release 80-100 ug dopamine daily, respectively.

Oral administration of 500 mg L-dopa to humans caused a fall in
plasma LH levels within one hour. The decline was never below the
physiological range, but it was suggested that the ovulatory LH surge
in women may be suppressed (Boden £3 31., 1972).

In conclusion, it seems that dopamine may have some inhibitory
function in controlling gonadotropin release. The earlier work showing
its stimulatory effect cannot, as yet, be ignored and perhaps it has a
dual role depending on endogenous endocrine conditions. In addition,
Ojeda and McCann (1973) obtained evidence that dopamine may be involved
with FSH but not LH release. They showed that pimozide (a dopamine re-
ceptor blocker) selectively lowered plasma FSH levels in the castrated
rat, while diethyl-dithiocarbamate (DDC), which lowers noradrenaline
by inhibiting the conversion of dopamine to noradrenaline, lowered
plasma LH. Correlated with this Choudhury 25 El: (1973) have shown
that activating dopamine receptors with apomorphine stimulated FSH

release.

37

2. Noradrenaline

 

All the results obtained after altering levels of nor-
adrenaline in the hypothalamus indicate that it is concerned in the
tonic and cyclic release of LH and that it acts as a stimulatory
transmitter.

When rats are unilaterally ovariectomized, the remaining ovary
undergoes compensatory hypertrophy due to an increase in gonadotropin
release because of the reduction in ovarian secretion and therefore in
negative feedback effects. Inhibition of catecholamine synthesis by a-
methyltyrosine prevented this compensatory growth in immature rats
(Mfiller g£_§1,, 1972) and selective destruction of noradrenergic nerve
terminals by 6-hydroxydopamine prevented this growth in adult rats (Zolo-
vick, 1972). The effect of a-methyltyrosine was reversed by both DOPA
and DOPS. These results indicate that noradrenaline is involved in the
tonic release of gonadotropin after unilateral castration. Similar
results were obtained in bilaterally castrate males Where administration
of either a-methyltyrosine or DDC lowered plasma LH levels. In this
preparation their effects were reversed by DOPS but not by DOPA (Ojeda
and McCann, 1973).

Additional evidence supporting the noradrenergic control of tonic
gonadotropin release was shown in rats in which the hypothalamus com-
pletely deafferentated. In these rats there was a fall in hypothalamic
noradrenaline levels to 38 percent of the normal concentration and at
the same time plasma and pituitary LH levels fell significantly, while
dopamine levels remained normal. When only the anterior portion of

the hypothalamus was deafferentated there was only a small decrease in

38
hypothalamic noradrenaline and only a slight fall in plasma LH (Blake
‘gtngl., 1972; Weiner ES 31., 1972b).

Noradrenaline may also be concerned with the cyclic release of the
gonadotropins in that Kalra and McCann (1973b) have shown that noradrena—
line may be the synaptic transmitter in the pathway between the preoptic
area and the median eminence, because the LH surge obtained after stimu-
lation of the preoptic area was reduced by a—methyltyrosine (which lowers
noradrenaline and dopamine levels in the hypothalamus) and by dopamine-
B-hydroxylase inhibitors (which lower noradrenaline levels only). The
LH surge in these animals was restored by DOPS, which selectively raises
noradrenaline levels but not by a dose of DOPA which raises mainly
dopamine levels. The LH surge after stimulation of the median eminence
was not affected by the synthesis inhibitors (Kalra and McCann, 1973b).
This type of experiment was also carried out in ovariectomized rats.

When these animals are primed with estrogen and then treated two days
later with a single injection of progesterone or estrogen there is a
surge of LH, which can be prevented by the synthesis inhibitors and can
be reversed by selectively raising noradrenaline levels, but not dopamine
levels (Taleisnik g£_al., 1970; Kalra gt 31., 1971, 1972; Kalra and
McCann, 1973a).

Craven and McDonald (1971) have shown that infusion of noradrenaline,
but not of dopamine, into the arcuate nucleus in rats pretreated with
a monoamine oxidase inhibitor can advance the critical period and
therefore the time of the LH surge before ovulation. On the other hand,
neither noradrenaline nor dopamine placed in the arcuate nucleus was
able to overcome the anti-ovulatory effect of pentobarbital or reserpine

(Craven and McDonald, 1971, 1973). They suggested that noradrenaline

39

may be involved in the changes occurring early on the day of proestrus
leading up to the LH surge, rather than in the release of LH itself.
In this connection, Kanematsu £5 31. (1972) found that intraventricular
administration of noradrenaline stimulated the release of LH in rabbits
but with a different onset and duration from that produced by coitus
or electrical stimulation, suggesting that while noradrenaline was in-
volved with LH release it was not via the same mechanisms as that pro-
ducing the ovulatory surge of LH.

3. Serotonin

Inhibitory Effects

 

Since the administration of 5-HT caused atrophy of the
reproductive organs and delayed puberty in immature mice, Robson and
Botros (1961) considered it likely that 5-HT was an inhibitor of gonado-
tropin release. This was supported by the findings of Vaughan E£.§Er
(1970) who showed that intraperitoneal administration of 5-HT prevented
the compensatory ovarian hypertrophy normally seen after unilateral
ovariectomy in rats. Subcutaneous or intraperitoneal injections of 5-HT
were also found to inhibit ovulation both in adult rats and immature
rats induced to ovulate with PMS (O'Steen, 1964, 1965; Endersby 33.31.,
1970; Labhsetwar, 1970; Time 25 al., 1973). Similar results were ob-
tained by raising levels of 5-HT in the body, including the brain, by
administering monoamine oxidase inhibitors; this was shown in the
hamster, rat and immature rat (Alleva gg al., 1966; Kordon gt 31,,

1968; Kordon, 1969; Labhsetwar, 1970).
The hypothesis that S-HT inhibits gonadotropin release, however
does not explain all the results. Most drugs that inhibit ovulation by

a central action do so by suppressing the hypothalamic Stimulation of

4O
pituitary that occurs just before the critical period. However, sub—
cutaneous administration of 5-HT had no effect just before the critical
period but only inhibited ovulation when given after the critical
period, or late in diestrus (Endersby and Wilson, 1973). The timing of
the inhibitory effects correlates well with the times in the cycle when
ovarian hormones are secreted and Wilson and McDonald (1973, 1974),
suggested that the anti-ovulatory effect of the 5-HT is due to a peri—
pheral vasoconstrictor action preventing the passage of the ovarian
steroids away from the ovary to the hypothalamus. The main support of
this hypothesis is that S-HT can antagonize the ovulatory action of
exogenous LH and that the anti-ovulatory effect of the 5-HT on sponta-
neous ovulation is reversed by a vasodilator (dipyradimole). A vaso-
dilator compound (apresoline) has also been shown to prevent the atrophy
of the reproductive organs in male rats caused by 5-HT (Boccabella g3
a1,, 1962). The experiments in which monoamine oxidase inhibitors
Showed an anti-ovulatory effect may be due to raised levels of peripheral
5-HT, although the authors correlate the effect with raised brain
levels.

In spite of all these findings there is still much evidence that
5-HT may be a central inhibitory transmitter. Firstly, there is the
presence of 5-HT nerve terminals in the hypothalamus, particularly the
suprachiasmatic nuelcus, retrochiasmatic area and the median eminence;
this latter area is capable of synthesizing S-HT from tryptophan and
this is known to occur only in tissues containing tryptaminergic
neurons (Andén gt 31., 1965; Hamon g£_§1,, 1970). Secondly, there is a
tryptaminergic tract from the midbrain to the hypothalamus (Ungerstedt,

1971; Fuxe g£_al,, 1968b) and electrochemical stimulation of the ventral

41
tegmentum and raphé in the midbrain, or tracts leading from these areas
to the hypothalamus, inhibits spontaneous ovulation in rats and also
reduces plasma LH levels. This inhibitory effect can be prevented by
lesioning the tracts between the two areas (Carrer and Taleisnik, 1970,
1972).

Changes in 5-HT concentrations and metabolism in the hypothalamus in
different endocrine states have been noted, although the work is not as
extensive as that on the catecholamines. In the intact ewe, 5-HT levels
in the median eminence fall significantly just before the LH surge
(Wheaton 35 al., 1972). When ovarian steroids were administered to
castrated rats so that they exerted a negative feedback effect on gonado-
tropin release, there was a rise in hypothalamic tryptophan levels
(Bapna gt_al,, 1971), and also in S-HT levels in the midbrain area
(which included the hypothalamic area in this particular work) (Tonge
and Greengrass, 1971). A Single injection of estradiol either on day
one or eleven of life raised 5-HT brain levels in immature males and
females (Guilian 25 31., 1973). Kato (1960) found a similar effect
after chronic administration of estradiol to immature rats. Conversely,
ovariectomy of neonatal females reduced brain 5-HT levels. As well as
brain S-HT being influenced by the steroids, the metabolism of the
steroids is altered by 5-HT. Pretreatment with 5-HTP increased the
accumulation of 3H-estradiol in the hypothalamus and pituitary after
intravenous injection of the labeled steroid (Kordon g£_al., 1972).
Treatment with progesterone may inhibit the synthesis of 5-HT, because
accumulation of 3H-tryptophan was reduced in castrated rats pretreated

with progesterone (Kordon and Glowinski, 1972).

42

Intravenous injection of 5—HT can inhibit ovulation in rabbits if
given just before the expected LH surge, indicating a central effect
(Currie gt 31., 1969) and intraventricular administration of 5-HT inhi-
bited LH and FSH release in both intact and castrated male and female
rats (Kamberi_gEHal., 1970b, 1971a; Kamberi, 1973; Schneider and McCann,
1970d), and caused atrophy of the gonads in immature rats (Corbin and
Schottelius, 1961). 5-HT had no effect on the pituitary itself (Kamberi
and McCann, 1969a,b).

Reports on the effect of intraventricular 5-HT on spontaneous
ovulation differ. When high doses of 5-HT (50-200 pg per rat) were in-
jected at various times of the day of proestrus, there was no effect on
ovulation (Rubinstein and Sawyer, 1970; Schneider and McCann, 1970d;
Wilson and McDonald, 1974). However, recently Kamberi (1973) found
that 1-5 ug of 5-HT injected intraventricularly just before the critical
period inhibited ovulation; it was ineffective when given by the intra—
cardiac route, while its precursor 5-HTP, which passes through the
blood-brain barrier, was effective. Intraventricular 5-HT also inhibited
the facilitatory effect of prOgesterone on induced ovulation (Zolovick
and Labhsetwar, 1973). Implants of a monoamine oxidase inhibitor
(nialamide) in the median eminence were also effective in blocking ovu-
lation, but this may be due to the compound itself and not the raised
S-HT levels (Kordon and Vassent, 1968).

Wilson (1974) injected 5-HT into the ventrolateral part of the
anterior hypothalamus, e.g., in the medial forebrain bundle, and showed
that it inhibited induced ovulation in immature rats. This suggests

that the tracts from the midbrain that Carrer and Taleisnik (1970) showed

43
could inhibit ovulation may be stimulated by the 5-HT injections and
this may be the site for inhibition of cyclic release.

In 1959, Brodie and Shore suggested that 5-HT and noradrenaline
were transmitters in Opposing systems, which were in dynamic balance.
Lippmann (1968) suggested that gonadotropin release was controlled, not
by individual levels of the amines, but their relative proportions, so
that when 5-HT levels were high compared to noradrenaline, or when nor-
adrenaline levels were 1ow compared to 5-HT, then gonadotropin release
was inhibited. Labhsetwar (1971) made a similar comment and showed that,
when both amines were reduced to the same extent, there was no effect
on gonadotropin release. He also showed that dopamine can reverse the
inhibitory effects of 5-HT on ovulation induced by PMS and progesterone
(Zolovick and Labhsetwar, 1973).

5-HT is a precursor of the pineal hormone, melatonin, which has
been shown to inhibit gonadotropin release and activity (Wurtman SE 31.,
1968; Debeljuk ggflal., 1970; Kamberi §£_§1,, 1971a) and also to inhibit
ovulation (Longenecker and Gallo, 1971; Ying and Greep, 1973) and delay
puberty (Collu £5 31., 1973). Conversely removal of the pineal can
induce ovulation (Mess SE 31., 1973). Martini and his coworkers
(1968) investigated the various pineal principles and noted that they
had a selective effect on LH and FSH release. They found that implants
of melatonin and S-hydroxytryptophol in the median eminence of castrated
male rats reduced pituitary and plasma LH but did not affect levels of
FSH. On the other hand, 5-HT and 5-methoxytryptophol implants only
reduced FSH levels in the pituitary. The pineal principles have a
diurnal rhythm; in the dark period melatonin concentration is high and

5—HT low and during the day the levels are reversed. Fraschini (1970)

44
suggested that this pineal rhythm controls the rhythm seen in pituitary
gonadotropins which peak daily in the afternoon. He suggested that
the pineal substances travel to the basal hypothalamus via the cerebro-
spinal fluid and then alter releasing hormone activity.

The Stimulatory_Effect of 5-HT

 

Although the majority of the results Show that S-HT acts
as an inhibitor of gonadotropin release, there is also some evidence
that 5-HT can stimulate both tonic and cyclic gonadotropin release.

Most of the evidence has been obtained after administration of p-chloro-
phenylalanine (PCPA), a compound known to inhibit synthesis of brain
5-HT by antagonizing the action of tryptOphan hydroxylase (Roe and
Weissman, 1968). It has been shown that PCPA prevents the onset of
puberty (Fajer ggflal., 1970) and the Sudden release of FSH normally seen
at puberty (Brown, 1971). It also reduces reproductive weights in hemi-
castrated male mice (Fawke and Brown, 1970) and reduces testosterone
levels in the plasma of intact male rats (Bliss 35 31., 1972). At the
same time PCPA raises pituitary FSH levels in intact and castrated male
rats (Brown and Fawke, 1972). All these results indicate that the re-
lease of FSH stores in the pituitary is inhibited after PCPA and there-
fore the gonads are not Stimulated to grow or secrete steroids. PCPA
also inhibits induced ovulation if given 20 hours before the critical
period (Kordon g£_§13, 1972), that is at a time stradiol secretion is
taking place, in order to eventually stimulate the release of the LH
surge. Kordon suggested that S-HT is necessary for the positive feedback
effect of the estradiol, perhaps by enhancing its uptake into particular

hypothalamic sites.

45

Complete deafferentation of the hypothalamus lowers S-HT levels
within this area by 70 percent, as well as significantly lowering nor—
adrenaline. It is possible that the reduction in gonadotropin release
seen after the operation is due to the reduction in tryptaminergic
activity (Weiner, 1973).

S-HT itself has been shown to stimulate gonadotropin release. For
instance, chronic subcutaneous administration of 5-HT for 12 weeks in-
creases spermatogenesis in adult male rats (Kinson and Lui, 1973);
intraocular or subcutaneous 5-HT induces ovulation in persistent estrous
rats (Takahashi SE El., 1973); a single intravenous injection of 5-HT
raises plasma LH levels in ovariectomized rats pretreated with ovarian
steroids (McCann 35.3l" 1960).

Interesting results have been reported on the immature animal
induced to ovulate with PMS. Brown (1967, 1967) showed that subcuta-
neous injections of 5-HT potentiated induced ovulation in immature mice,
while anti-S-HT compounds, such as LSD and methylsergide, inhibited
ovulation. Similar results were obtained in immature rats treated with
PMS. Thirty-day old rats underweight for their age did not ovulate
after PMS treatment, but if 100 mg/kg of 5-HT was given subcutaneously,
just before critical period, then 50 percent ovulated (Jaitley SE 31.,
1967). One mg/kg of 5-HT intraventricularly administered also induced
ovulation in both immature and underweight rats. The stimulatory site
of action of 5-HT appears to be the paraventricular nucleus, as injec-
tions of 2 pg per rat Specifically into this nucleus Stimulated ovulation
in 77 percent of underweight immature rats; injections into the arcuate
nucleus, the suprachiasmatic nucleus and the atnerior hypothalamus were

ineffective (Wilson and MacDonald, 1973). In adult rats, however,

46
the suprachiasmatic nucleus may be the stimulatory site, as this nucleus
contains high levels of 5-HT, and while lesions in this region pre-
vented ovulation (Artnes-Rodrigues and McCann, 1967), stimulation of
this nucleus induced ovulation (Critchlow, 1958).

As mentioned previously, Fraschini §£_§1, (1971) suggested that
the circadian rhythm of gonadotropin release is controlled by the pineal
principles. Quay (1963) has shown 5-HT levels in the pineal are at
their highest daily at midday and higher on the day of proestrus, than
on other days. He also showed that hypothalamic levels of 5-HT have a
circadian rhythm with a peak each day in the late afternoon, at the
expected time of the critical period, and when pituitary levels of the
gonadotropins are at their highest. It is possible, therefore, that
S-HT is involved in the control of the release of the ovulatory surge
of LH (Quay, 1968).

A most interesting finding by Ladosky and Gaziri (1970) and also
confirmed by Guilian £3 31, (1973) is that 5-HT may be involved in
sexual differentiation. They found that between days 10-14 of life,
there is a rise in brain levels of 5-HT in females which can be pre-
vented by administration of testosterone, or by ovariectomy on day one
of life. Hardin (1973a) obtained rather different results in that the
5-HT levels in the females were higher only on day two and not later in
life. Injections of estradiol in the neonatal period can raise S-HT
levels in both male and female rats (Kato, 1960). The alteration in
S-HT levels may be due to the effect of the steroids on monoamine oxi-
dase (MAO) activity; for instance, activity on day 12 is reduced by
testosterone given on day one of life and raised after castration

(Ladosky and Gaziri, 1970; Gaziri and Ladosky, 1973). On the other

47
hand, the differences may be due to the higher levels of 5-HTP decar-
boxylase found in 2-day-old females compared to their male litter mates
(Hardin, 1973b).

Conversely, Vaughan g£_§1, (1969) have shown that early administra-
tion of 5-HT can antagonize the androgenizing effects of neonatal testos—
terone in the female. This would indicate that the prevention of the
cyclic release of gonadotropins by neonatal androgens, as seen in the
normal male or after exogenous administration of testosterone to the
female occurs via raising MAO levels and thus reducing 5-HT levels in
the brain. These findings can be correlated with some fluorescent
studies, which showed that when sexual differentiation is prevented in
males by neonatal castration or the administration of an anti-androgen
(cyproterone acetate) there was an increase in fluorescence in most
hypothalamic nuclei due to raised amine levels (Hyppa and Rinne, 1971).

C. Gonadal Steroid Feedback in Rggulatigg_Gonadotropin Release

1. .Nggative Feedback

When the ovaries or testes are removed in the rat, both
plasma and pituitary levels of LH show a marked rise (Gay and Midgley,
1969 and Yamamoto 35 31., 1970). In the case of FSH, the situation
appears to be quite similar, the only difference being that the eleva-
tion in plasma levels of FSH is less dramatic than the elevation of LH.
Also, repeated sampling and measurement by radioimmunoassay have re-
vealed oscillations in the castrate rat (Gay and Midgley, 1969) and
rhesus monkey (Dierschke £5 31,, 1970) which have been termed circhoral
fluctuations. Probably these represent a periodic pulsatile release

of gonadotropin-releasing factors into the porta1 vessels. This effect

48
of ovariectomy could be inhibited by the administration of estrogen or
progesterone (Ramirez 25 21., 1964; Smith and Davidson, 1974). The
effects can be noted within one day and become maximal after two or
three days. The sensitivity to the inhibitory effect of estrogen on
gonadotropin release appears to be somewhat less in castrate males
(Kalra SE 31., 1971).

2. Positive Feedback

 

Estrogen and administration to regular cycling rats during
diestrus has been shown to advance the time of ovulation (Everett, 1948;
Brown-Grant, 1969; Weick g£_§1,, 1971). This effect can be blocked as
well as ovulation itself by estrogen antibody administration (Ferin SE
.31., 1969, 1974; Knobil gt 31., 1974). Estrogen administration to long
term ovariectomized rats causes a daily LH surge (Legan and Karsch,
1975).

Progesterone administration by itself, inhibits LH secretion in
early diestrus (Everett, 1948) and interferes with the LHRH induced LH
release (Martin g£_§1,, 1974). However, if progesterone is administered
in an estrogen environment, such as is present in proestrus, an LH surge
can be stimulated (Everett, 1948; Zeremaker, 1966; Brown-Grant and
Naftolin, 1972). This facilitatory effect of progesterone upon estrogen
action is seen when progesterone is given to estrogen primed, long term
ovariectomized rats. Under these conditions an LH surge is seen which
is Similar in magnitude and timing of the preovulatory LH surge (Cal-
garis £5 31,, 1971). In addition, surges of FSH and Prl accompanied
this LH surge correlating it with those changes occurring during pro-
estrus (Calgaris gt 21., 1974). Recent studies have shown daily morning

progesterone peaks during the estrous cycle (Mann and Barraclough, 1973)

49
and a large progesterone surge on the morning of proestrus (Kalra and
Kalra, 1974). Thus, a synergistic interaction between progesterone
and estrogen may normally occur during the estrous cycle.

Under the influence of FSH (and a low basal level of LH), the
ovarian follicles mature and at a certain stage secrete estradiol
(Schwartz, 1969; Ely and Schwartz, 1971). Following a peak level of
plasma estradiol at around midday (Shaikh, 1971) on the day before ovula-
tion, there is a sudden surge of LH, FSH and prolactin from the pituitary
in the late afternoon on the day before ovulation, lasting two to four
hours (Naftolin SE 23., 1972; Mahesh and Goldman, 1971; Freeman 33.33,,
1972). The surge of LH (but probably not of FSH or porlactin) is
necessary for ovulation to take place. It initiates (l) the secretion of
progesterone from the follicles and/or interstitial cells (Leavitt 33
33,, 1971) and (2) changes in the follicle walls so that the follicles
rupture about 12 hours later, each follicle releasing an ovum. Luteal
tissue then grows to fill the follicles to form corpora lutea which are
capable of producing progesterone, although they do not secrete high
levels unless fertilization takes place.

In the rat the stimulation of LH release by the rise in plasma
estradiol can only occur during a critical period of two hours during
the afternoon of the same day. The exact time is controlled by the time
of commencement of the light period for that day. For example, on a
fixed regime of 14 hours of light starting at 0600 h, the critical
period occurs between 1400 and 1600 h (Everett 35.33,, 1949). The actual
release of LH into the blood after stimulation occurs between 1200 and

2000 h with a peak at 1800 h.

50

The rise in progesterone levels occurring simultaneously, or just
after the LH surge, is stimulated by the LH (Barraclough 3£_3l,, 1971;
Piacsek 3£_3l,, 1971) and only declines slowly over the following dark
period. It is thought to be necessary for inducing sexual receptivity
(Boling and Blandeau, 1939) in the rat and may also help control the
duration of the LH surge (Kobayashi 3£_3l,, 1970). In the rat, all
these changes occur over a period of four or five days (the estrous
cycle).

In the human, an analogous series of change occur over a period of
approximately 28 days (the menstrual cycle). During the first half of
the cycle (or follicular phase) low levels of FSH and LH stimulate the
ovarian follicles to secrete estrogen which reaches a peak concentra-
tion just before mid-cycle, and then, either on the same day or one day
later, there is a dramatic increase in LH secretion (and to a lesser
extent FSH) which lasts one or two days. This is equivalent to the
LH surge in the rat and ovulation takes place at some time towards the
end of this mid-cycle period. After ovulation the corpus luteum formed
from the ruptured follicle secretes progesterone and also some estrogen
in the second half of the cycle (the luteal phase) near the time of im—
plantation. If fertilization does not occur, the corpus luteum re-
gresses approximately two weeks after its formation and the steroid
secretion falls to zero (Ross 3£H3l., 1970; Vande Weile 33.33., 1970).

3. _Hypothalamic Sites of Feedback
The sites of steroidal feedback effects in the hypotha-
lamus have been the object of much work. It is now considered that

there is a tonic center in the arcuate nucleus and median eminence area

51

which controls low tonic secretion of gonadotropin necessary for main-
taining gonadal weight and function. In the female there is an addi-
tional center in the preoptic area, which controls the cyclic release
of LH just before ovulation (Gorski, 1966). It is thought that the
cyclic center sends pulses at a regular time each afternoon to stimulate
the tonic center in the median eminence (Tejasen and Everett, 1967;
Gorski, 1968), but the latter only responds on the day before ovulation
when the estradiol levels are high. It is suggested that the estradiol
lowers the threshold of Stimulation of the median eminence (McDonald
and Gilmore, 1971; Sawyer and Hilliard, 1971). Everett (1964) has
shown that there is a neural pathway connecting the two centers. Stumpf
(1968) demonstrated by autoradiography that estradiol is taken up in
significant quantities in the anterior hypothalamus, especially in the
preoptic and median eminence areas, and Kato (1973) showed that there
are significantly higher concentrations of estrogen receptors in these
areas. Implants of estradiol are more effective in inducing gonado-
tropin release when placed in the median eminence, although implants

in the preoptic area are also active in some situations (Smith and
Davidson, 1967; Davidson, 1969). The site of the negative feedback
effect of the steroids also appears to be at the median eminence

levels (Chowers and McCann, 1967; Smith and Davidson, 1968; Taleisnik
3£H3l., 1970). Possibly the steroids have biphasic effects on the
threshold of stimulation of the median eminence, first lowering and

then raising it (Beyer and Sawyer, 1969).

52

IV. The Effect of Light Upon Reproductive Function

 

A. General

1. Light as an Environmental Constituent

 

Each human organism receives a finite number of inputs
from the world beyond its integument. These inputs, collectively termed
"environment", include chemical, physical, biological, and informational
factors; they presumably exerted the major influence on man's develop—
ment throughout his evolutionary history, and continue to affect him
now.

Solar radiation constitutes a ubiquitous and essential component of
man's environment. Besides serving as the ultimate source of his food
and energy, it has also acted directly upon man to alter his chemical
composition, control the rate of his maturation, and drive or entrain
his biological rhythms.

2. Characteristics of Natural Lighting

 

The spectral composition of sunlight at the earth's sur—
face approximates that of the white light emitted by a theoretical
black body heated to about 5600°K, minus the ultraviolet radiation
below 290 nm that is unable to penetrate the ozone layer and atmospheric
shield surrounding the earth. Hence the solar spectrum is continuous
and the relative intensities of any of its component visible wave-
lengths do not differ by more than two-fold; the ratios of the radiant
fluxes of red, blue, and yellow bands contained in the white light of
the typical midday sun approximate 1:1:1.

Incandescent light sources emit spectra that approximate those of
heated black bodies. Their color temperature (the termpature to which

a theoretical black body would have to be heated in order to emit a

53
comparable spectrum) is considerably lower than that of sunlight, hence
a larger fraction (about 90%) of their total radiant power consists of
infrared radiation that provides heat rather than light (Thorington
33H3l., 1971). Within the visible portions of incandescent spectra,
the relative fluxes at different wavelengths are, as expected, maximum
in the red and minimum in the blue. The identity of those wave lengths
of light that are most important for the reproductive responses to light
has not been established for the mammal. Allardyce 95.2l: (1942)
studied the fertility rate of rats exposed to different colors of light
and found that most young were born in yellow light and none were born
in blue light; the effects of red light and green light were interme-
diate. In the duck, Benoit and Assenmacher (1966) found that yellow
light directed at the eyes did not cause gonadal stimulation but that
red light was strongly stimulatory. As pointed out by Wurtman (1967),
sunlight and both incandescent and fluorescent artifical lighting emit
a range of wavelengths much broader than that perceived by mammalian
photoreceptors, so that the quality of a light source has not been an
important issue. However, certain types of fluorescent lighing that
parallel more closely the emission pattern of sunlight have been shown
to be more stimulating to the gonads than the usual cool-white fluorescent
bulbs (Wurtman and Weisel, 1969) and thus the light source should be
identified in reports of light effects.
B. Effects of Photoperiod on Rgproductive Function
The system upon which light acts involves ultimately the
gonads, which are producing the gametes and also the gonadal steroids.
The gonads are stimulated to grow and secrete by gonadotropic hormones

from the anterior pituitary gland. The pituitary synthesizes and

54
releases its gonadotrOpic hormones under the influence of neurohormones
produced in the hypothalamus and released into the hypothalamic-hypo-
physial system in the region of the median eminence. The neurohormones
may stimulate or inhibit synthesis or release of the gonadotropins from
the pituitary. Although there is some evidence for a direct effect of
certain treatments on the gonads, for the most part whatever acts on the
reproductive system acts through an influence on the pituitary itself or
on the hypothalamic control centers. The first problem then is to trace
the pathway of the light stimulus from its external source to the hypo-
thalamus.

1. Pathways of LiEEE Impulses Affectinngeproduction

 

In adult mammals the receptor for light is the retina of
the eye, even though measurable light does penetrate the skull (Ganong
.E£Hél°’ 1963). Whether light is excluded by covering the whole head
with a hood (Bissonette, 1936), by severing the optic nerve (Bissonette,
1938), by destroying the retina (Thomson, 1951), or by removing the eye
(Corbin and Daniels, 1969; Hoffmann, 1967; Hoffmann 3£H3;., 1968) the
result is the same; treated animals do not respond to altered or added
external lighting. Lisk and Kannwischer (1964) have reported that im-
plantation of glass fibers into the hypothalamus so that light could be
conducted there from the outside caused some increase in ovarian weight
in blinded animals exposed to constant light, compared to blinded
animals in which the glass fiber was Shielded. However, these experi-
ments have not been purused further, and it is possible that energy
in any form would have a stimulatory effect when conducted to appro-

priate areas of the hypothalamus.

55

Impulses initiated by light impinging on the retina are conducted
back toward the brain via the optic nerves, which then join briefly at
the optic chiasm. Some of the fibers cross there, and the majority Of
the fibers continue back toward the lateral geniculate bodies in bundles
known as the optic tracts. However, sOme crossed fibers, which form the
inferior accessory optic tract, leave the primary optic tracts imme-
diately behind the chiasm and join the median forebrain bundles to termi-
nate in the medial terminal nucleus of the midbrain (Hayhow, 1960).
These accessory fibers have proven to be the most important fibers for
reproductive responses to light. Destruction Of the inferior accessory
optic tract blocks the effect of added light on reproduction, whereas
total destruction Of the primary Optic tracts does not (Critchlow and
DeGrOOt, 1960).

Beyond this point the pathway has been established no further. That
is not to say that other areas of the brain have not been implicated
in responses to light, but only that clear-cut neuron-to-neuron pathways
have not been traced.

Information about light ultimately reaches some center in the hypo-
thalamus, where it is probably integrated with other information related
to reproduction and translated into changes in amount and nature of
neurohormones released to the pituitary.

Between the medial terminal nucleus of the midbrain and the hypo-
thalamus the light information undoubtedly follows many pathways, and
the information is probably used in different ways by many systems. One
loop in this circuit that has been identified involves the pineal gland.
Although the actual role Of the pineal in reproduction is not yet clear,
it does show morphological and functional changes in response to light,

which suggest that it may mediate some of the effects of light on

56
reproduction. More detailed discussions of the pineal can be found in
recent reviews (Quay, 1969, 1970; Reiter and Fraschini, 1969; Reiter and
Sorrentino, 1970).

2. Correlation with Reproductive Activity

 

Bissonette (1932) in one of the earliest demonstrations of
the stimulatory effects of light, showed that female ferrets came into
heat in January instead of March, if they were exposed to about six
hours of extra light per day in November and December. Subsequently,
other workers have shown this stimulatory effect of increased photoperiod
in the horse (Burkhardt, 1947), mink (Enders and Enders, 1963), racoon
(Bissonette, 1937), cat (Scott, 1959), and vole (Clark and Kennedy, 1967).
Conversely, keeping female ferrets in short daily photoperiods can delay
the onset of estrus (VanDerWerfften Bosch, 1963). As might be expected,
such species as the sheep, and goat, which normally exhibit estrus in
the Fall, have been shown to advance their sexual activities when exposed
to artificially shortened days and to go into anestrus earlier if photo-
period is increased in the Fall (Bissonette, 1941; Hafez, 1951).

Alternating light and dark is sometimes more stimulatory than con-
tinuous light. In the ferret, for example, exposure to 14 or 16 hours
of light per day has a greater stimulatory effect than does constant
exposure to light (Donovan, 1967; Hammond, 1951). This led Hammond
(1951) to conclude that dark played an important role.

Some species, including the domestic rabbit (Clegg and Ganong,
1969) the cow (Asdell, 1964), and the pig (Clegg and Ganong, 1969) have
been domesticated for so long that they seem to have escaped from the
seasonal variations in breeding activity. Seasonal variations in fer—

tility remain in most of these Species (Clark and Kennedy, 1967) but

57
breeding season is no longer well defined. Laboratory animals also tend
to show continuous cycles in the unvarying lighting conditions of the
animal quarters; hamster (Orsini, 1961), rat (Everett, 1961), and mouse
(Bingel and Schwartz, 1969).

3. Effect on Reproductive Cycle Length

 

The length of the reproductive cycle in the rat can some-
times be altered by the length of the photoperiod. Rats exposed to 12
hours of light and hours of darkness (12L:12D) show four day cycles
while rats exposed tO a (14L:10D) or (16L:8D) regimen show predominantly
five day cycles (Hoffmann, 1968, 1969). In 24 hours of light per day
(LL), the animals eventually go into persistent estrus. Differences
in sensitivity of day length are apparent among various strains of rats
(Hoffmann, 1970).

4. Effect on Sexual Maturation

 

An effect of photoperiod on the time of occurence of
sexual maturation has been frequently demonstrated. Exposing immature
female rats to constant light advances vaginal Opening (Fiske, 1939;
1941; Moszkowska and DesGouttes, 1962; Piaseck and Hautzinger, 1974),
and light deprivation by blinding or exposure to constant dark delays
it (Fiske, 1939, 1941; Truscott, 1944). Constant light decreased the
age at which large ovulatory follicles appeared therefore facilitating
GnRH induced Ovulation at an earlier age (Steger 33.33,, 1975).

5. Effect on Timing of Ovulation and Mating Behavior

 

Probably the first indication that estrus and ovulation
occurred at a specific time of day related to the light—dark cycle was
the report by Dempsey 33,33. (1934). They found that behavioral estrus

in guinea pigs occurred only in darkness and that the time of estrus

58

tended to shift as day length varied, so that the occurrence of estrus
remained in the dark period. Subsequently, Hemmingsen and Krarup (1937)
did a similar study in the rat. As in the guinea pig, estrus in the rat
occurred during the dark, and a complete reversal of the light-dark
schedule resulted in a reversal in the time of onset Of estrus within a
few cycles. In a similar experiment with the mouse, Snell 3£_33, (1966)
not only found that mating behavior predictably occurred at a particular
time of day but also that ovulation occurred between midnight and 0400 h.
Later, Everett, and Sawyer (1949, 1950) found that the release of ovu-
lating hormone in the rat could be checked by barbiturates but only
during a limited period of time--between 1400 and 1600 h on the day of
proestrus (lights on at 0500 h and off at 1900 h; 14L:10D). As long as
the time is measured from the midpoint of dark, the critical period
for ovulation blockade occurs between 1400 and 1600 h, whether the length
of the daily photoperiod is 12, 14 or 16 hours. Thus, the onset of the
critical period and the resulting ovulation seem to be associated with
the midpoint of dark or light, rather than with the beginning of dark
or light (Alleva 3£_3l,, 1970; Hoffmann, 1969). When the light to dark
ratio is held constant but the photoperiod is shifted relative to solar
time, the time of maximum ovulation blockade (Everett, 1952; Everett
and Tajasen, 1967) and the time Of ovulation (Alleva 33 33., 1970;
Austin and Braden, 1954; Carlysle and Carter, 1961) shift a correspon-
ding number of hours.

Psychoyos (1966) has suggested that the lighting schedule can also
affect the sensitivity of the ovary to LH. He gave PMS to 28-day-Old
rats, placed them in conditions varying from 0 to 24 hours of light per

day, and administered exogenous LH 2 days later. All the animals

59

ovulated, but the 18L:6D group shed twice as many ova, when compared to
all the others.
6. Effect on Luteal Phase of the Reproductive Cycle

Little, if any, data are available to suggest that the
length of the luteal phase in infertile cycles is affected in any way by
light. The length of pregnancy, however, can sometimes be altered by
light. In most cases the reason for the effect is not yet known. In
the mink, delayed implantation is quite common, and females mating early
in the season tend to have a longer delay and therefore a longer total
gestation than those breeding later. Furthermore, increasing the photo-
period to which mink are exposed can cause earlier implantation (Pearson
and Enders, 1944). This is also true of the marten and the sable (Far-
ner, 1961). The exact mechanism of the delay in these species is not
clear. In the rat delayed implantation is seen during lactation, and
implantation can be induced by giving a single injection of estrogen
(Krehbiel, 1941); however, no hormonal regimen has been successful in
inducing implantation in the wild species that normally show the delay
(Canivenc and Bonnin-Laffarque, 1963; Hammond, 1951). All that can be
said is that the corpora lutea remain in a state of quiescence until
some external signal or internal clock turns them on.

The total length of gestation in the horse is known to vary with
the season. Mares delivering in winter have a gestation period as much
as 20 days shorter than mares delivering in spring (Asdell, 1964).
Whether this is due to a difference in time of implantation or to rate

of development of the foal is not known.

60

C. Effect of Constant Light or Constant Dark on Reproduction

 

In the complete absence of a light-dark alternation--in constant
light or constant dark--reproductive cycles may continue undisturbed or
may be profoundly altered. The rat and mouse go into a state or per-
sistent estrus fairly soon after exposure to constant light (mouse, Chu,
1965; Murthy and Russfield, 1970; rat, Browman, 1937; Everett, 1939, 1942;
Hardy, 1970; Hoffmann, 1970; Jochle, 1956; Lawton and Schwartz, 1965,
1967; Takahashi and Suzuki, 1969). Follicles develop to the preovulatory
stage and secrete estrogen, but ovulation does not occur, so that after
a time the ovary contains no corpora lutea. Only the follicles in various
stages of development are seen, and the constant secretion of estrogen
causes a continuous vaginal cornification, a high uterine weight, and
almost continuous sexual receptivity. If, instead of constant light,
the animals are in constant dark (by being housed in lightless quarters
or by blinding), the picture is entirely different. Browman (1937)
reported that rats kept in continuous dark showed normal estrous cycles,
but he apparently kept them in these conditions for only a few weeks.
J6ch1e (1956) kept animals in constant dark for 80 days and reported
that their cycles became longer, with more diestrus smears. Hoffmann
(1967) found that 20 percent of female Wistar rats blinded or placed
in continuous darkness as adults went into complete anestrus. Those
animals showed continuously leukocytic smears and at autopsy had very
small ovarian and uterin eweights, no recent corpora lutea, and only
small follicles in the ovaries. Females that were blinded at 21 days
of age, on the other hand, showed only a slight delay in reaching
puberty and later showed completely normal estrous cycles and organ

weights. It is important to note that light deprivation can have

61
quite different effects depending on the origin of the rats under study
and the age of the animals when treatment beings (Hoffmann, 1970).

In the hamster, constant light was first reported to have no effect
on the estrous cycle (Greenwald, 1963). However, the animals were
studied for only 60-70 days (16-18 cycles). When exposure was continued
for 6-14 months, hamsters also showed persistent estrus and failure of
ovluation (Kent 35 31., 1968). In constant dark, lL:23D or after
blinding, male and female hamsters show a very severe atrOphy of repro-
ductive organs (Reiter, 1974). This greater sensitivity to light depri-
vation in comparison to the rat is perhaps a reflection of the more
recent domestication of the golden hamster or perhaps due to the fact
that the hamsters studied have been pigmented animals, whereas most of
the rats studied have been albino strains.

Relatively little information is available on the effects of con—
stant light or dark in other species. Dempsey $5.31. (1934) reported
that guinea pigs continued to cycle in constant dark and reproduced
normally, although the onset of heat no longer could be correlated with
a specific time of day. Terry and Meites (1951) reported that sheep
kept in constant light for 7 weeks in the summer continued to cycle.
Waddill g£_al, (1968) exposed young pigs to constant light for one
complete estrous cycle and found no difference in ovulation rate.

In the experiments of Mennin and Gorski (1974), constant light
exposed ovariectomized rats did not respond to the positive feedback
action of estrogen and showed a different response to progesterone
priming than LD ovariectomized rats in terms of their serum LH release.

Constant light exposure may act by increasing serum levels of FSH

(Fink, 1975) or by increasing estrogen levels (Negro-Vilar 35 31.,

62
1968). The effect of estrogen in increasing the pituitary response to
GnRH has been demonstrated in zigg_and in zi££g_(Libertun ££H§£., 1974;
Arimura and Schally, 1971). The fact that estradiol benzoate accele—
rates vaginal opening more effectively in LL than LD rats (Piaseck and
Struer, 1975) suggests that the LL female is more sensitive to estrogen
than the LD female. However, constant light inhibited the ovulatory
response to PMS in the immature rat (Steger 35 31., 1975) suggesting
that LL alters the ability of the hypothalamus to regulate endogenous
GnRH injection. In a later study, Steger gt 31. (1976) have shown that
LL enhanced the pituitary's LH response to synthetic GnRH in the imma-
ture rat. However, in the mature female rat (Smith and Davidson, 1974)
an ovulation stimulus such as mating does not result in high LH release
due to a sensitivity of the pituitary to LHRH.

Even though female rats show continual vaginal cornification and
cease ovulation a few weeks after they are exposed to continuous light,
this condition is not irreversible. Takahashi g£_al, (1977) found
that when these animals were replaced in the darkness for ten hours,

80 percent of the animals ovulated approximately 46 hours later.
Furthermore, according to Brown-Grant (1974) light induced persistent
estrous rats show an extensive loss of photoreceptors but nevertheless
resume regular ovarian cycles when returned to a light-dark regimen.
Apparently retinal changes are not necessary to cause persistent estrus.
Pigmented rats may become anovulatory without any retinal changes at
the light microscopic level. Lambert (1975) has found that constant
light intensities of 5 or 25 uw/cm2 significantly prolonged estrus but
at these levels of LL no retinal pathology was apparent with the light

microscope in an albino strain of rat. Both white LL and red LL

63
prolong vaginal cornification at 100 uw/cm2 but only white LL produces
degeneration of retinal photoreceptors.

There are other changes in the hypothalamo-hypophysial-gonadal axis
which occur in response to constant light exposure. Fink (1974) reported
that the pituitary of LL eXposed rats was just as sensitive as that of
the LD exposed rat on the morning of proestrus in terms of LH response
to a single injection of synthetic GnRH.

The generalization has frequently been made that many if not all
effects of light upon reproductive processes occur through changes in
the release of various pineal substances which in turn affect the
homeostasis of the hypothalamo—hypophysial-gonadal axis. In the pheno-
menon of light induced persistent estrus in the rat, this is probably
not the case (Reiter, personal communication). Light itself creates
changes in the functioning of the neuroendocrine system that are inde-
pendent of involvement of the pineal gland (Ifft, 1962). Light may
induce a deficiency of catecholamines and an excess of serotonin in
hypothalamic areas a shypothesized by Kledzik and Meites (1974) in

light light persistent estrous female rats.

MATERIALS AND METHODS

I. Animals, Treatments and Blood Collection

 

Mature female rats used in these studies were obtained from Spartan
Research Animals, Haslett, MI. Animals were housed in light 14 h on,
10 h off (14L:10D) and temperature (25°:1°C) controlled rooms and pro-
vided with Purina Rat Chow (Ralston Purina Co., St. Louis, MO) and tap
water ad libitum. For the induction of persistent estrus, a group of
female rats was kept under constant illumination with an intensity of
10-15 foot candles as measured at floow level of plastic cages (Weston,
Model 915 light meter). After at least 5 weeks of constant light expo—
sure, those animals showing 10 consecutive days of vaginal cornifica—
tion were considered to be in persistent estrus and selected for these
experiments. Animals kept under 14L:10D conditions which showed regular
4-day cycles were selected on the day of proestrus unless otherwise
indicated. .

Blood samples were taken by decapitation or cardiac puncture under
light ether anesthesia. Blood samples were stored at 4°il°C for 24 h
to allow clot formation and serum was separated by centrifugation and
stored at —20°C until assayed for hormone concentration.

Alpha-methyl-para-tyrosine methyl ester HCl (Regis Chemical Co.,
Morton Grove, IL) and pargyline hydrochloride (Sigma Chemical Co.,
St. Louis, M0) were dissolved in 0.85% NaCl just before use. Estradiol

benzoate (EB) and progesterone (P) (Nutritional Biochemicals Corp.,

64

65
Cleveland, OH) were dissolved in corn oil. Synthetic gonadotropin re-

leasing hormone (GnRH, Abbot, Chicago, IL) was dissolved in 0.85% NaCl.

II. Radioimmunoassay of Serum Hormones

 

Serum concentrations of luteinizing hormone (LH), follicle stimu-
lating hormone (FSH) and prolactin (Prl) were determined using standard
double antibody radioimmunoassay procedures. Serum prolactin was
assayed using the method of Niswender at El- (1969), while serum LH and
FSH were determined by the methods described in the NIAMDD kits. Hormone
concentrations are expressed in terms of the standard reference prepara—
tions NIAMDD rat-prolactin-RP-l, NIAMDD rat -LH-RP-l, and NIAMDD rat-FSH-
RP—l. All serum samples were assayed in duplicate. Samples from indi-
vidual experiments were all tested in the same assay to avoid inter-
assay variability.

Methods used for ether extraction of plasma estradiol, progesterone
and testosterone, separation of bound from free steroids by charcoal
dextran and scintillation counting were previously described by Campbell
35 a}, (1977). Anti-progesterone-ll-BSA, GDN #337, 1:2500; anti-estra-
diol-6-BSA, GDN #244, l:20,000, and anti—testosterone-ll-BSA, GDN #250,
l:40,000; were provided through the courtesy of Dr. G.D. Niswender
of Colorado State University. Specificity of the estradiol anti-serum

was determined by Campbell 25 31. (1977).

III. Assay of Hypothalamis Luteinizing,Releasing;Hormone (LHRH)
A. Isolation and Preparation of Brain Tissue
Rats were decapitated and their brains immediately removed
from the cranium. A block of hypothalamic tissue constituting the

region lying between the rostral borders of the optic chiasm and

66
mammillary bodies and medial from the optic tracts were dissected to a
depth of about 2 mm. This was considered to be the medial basal hypo-
thalamus (MBH). Another block of tissue anterior to the MBH along the
same lateral borders and posterior to the anterior commisure was
dissected and considered to be the preoptic-anterior hypothalamic area
(AHA) (Figure l). The average weight of each of these hypothalamic
pieces was about 19 mg. Samples were stored on dry ice following their
dissection and then weighed and homogenized in 1 ml of 0.1 N HCl and
neutralized with 1 ml of 0.1 N NaOH. After centrifugation at 5000 g
for 20 minutes in a Sorvall refrigerated centrifuge at 4°C, the super—
natants were diluted with 0.1% gelatin in a phosphate-buffered saline
(PBS) to an appropriate concentration. Both serum and hypothalamic
extracts were stored at -20°C until assay.

B. Radioimmunoassay of LHRH

 

Hypothalamic LHRH was measured by the double antibody RIA
described by Nett 23 31. (1973). Anti-GnRH serum, R-42 pool, was pro-
vided by Dr. G.D. Niswender (Colorado State University), and was used at
a final dilution of 1:280,000. Synthetic LHRH (Lot 0, CN-79, 479-11K,
TM 10455 x 151-2, Parke-Davis Co., Detroit, MI) was used as a reference
preparation.

IV. Assay of Dopamine (DA), Norepinephrine (NE), and Serotonin (S-HT)
in Brain Tissue

 

 

A. Isolation and Preparation of Brain Tissue

 

Following decapitation, brains were removed and placed on
ice. The medial basal hypothalamus (MBH) and preoptic anterior hypo-

thalamic area (AHA) was dissected and frozen on dry ice. Tissue samples

67

were then weighed and homogenized in 20 ul of 0.4 N perchloric acid
(containing 10 mg EGTA/lOO ml). The MBH used in this study corresponded
roughly to that described in Materials and Methods, section III. Cuts
were made at the hypothalamic sulci, the mammillary bodies and caudal

to the optic chiasm. The AHA included the area medial to the hypo-
thalamic sulci, and caudal from the anterior commisure to the MBH
(Figure l).

B. Radioenzymatic Assay of Catecholamines

 

Dopamine (DA) and norepinephrine (NE) were assayed by the
radioenzymatic method of Coyle and Henry (1973) using catechol-O-methyl-
transferase (COMT) isolated from the rat liver by the method of Nikodi-
jevic £3 31, (1970). The assay was sensitive to 320 pg DA and 500 pg NE
and linear to at least 4 ng for both catecholamines. The presence of
tissue has been reported to reduce the activity of COMT (Coyle and
Henry, 1973). Since samples of hypothalami were all diluted to the same
concentration (1 mg/lO ul), values were not corrected for tissue inhibi-
tion. Results are expressed as ng DA or NE per gram wet weight.

Normetanephrine and methoxytyramine were separated utilizing the
solvent extraction and thin layer chromatography method of Ben-Jonathan
and Porter (1976). Amine content of samples were determined after
separation by counting chromatographic spots containing the 3H-labeled
metabolites in glass scintillation vials containing 10 ml of Scintiverse
(Fisher Scientific Products, Livonia, MI). Samples were counted in a
New England Nuclear, Mark II, scintillation counter.

C. Radioenzymatic Assay of Serotonin

 

Hypothalamic 5-HT concentrations were assayed according to the

method of Saavedra 35 31° (1973). Tissue samples were homogenized in

68

100 pl 0.1 N HCl (containing 10 mg EGTA/lOO ml). Rat liver N—acetyl
transferase was prepared by the method of Weissbach 33 21, (1961).
Hydroxyindole-O-methyl transferase extracted from bovine pineals (Pel-
Freez Biologicals, Inc., Rogers, ARK), was prepared by the method of
Axelrod and Weissbach (1961). Results are expressed as ng 5-HT per
gram wet weight.

Data were analyzed statistically by analysis of variance and the
Student-Neuman Keuls multiple range test (Sokal and Rohlf, 1969).

The level of significance chosen was P<0.05.

69

 

 

 

 

 

 

 

Figure l. DeGroot Atlas Section showing PreOptic-Anterior Hypo-
thalamic Area (AHA) and Medial Basal Hypothalamus (MBH).

Tissue sample

weight was 18:2.4 mg (AHA) and 2011.8 mg
(MGR) (N-54).

EXPERIMENTAL

I. Effect of Constant Light on Morning_and Afternoon Serum Levels of
Luteinizing Hormone (LH), Follicle Stimulatinngormone (FSH) and
Prolactin (Prl) in the Mature Female Rat

 

 

 

A. Objective

Critchlow (1963) postulated that exposure of rats to constant
illumination blocks the cyclic mobilization of LH essential for ovula-
tion, and that the retained follicles probably are responsible for the
continuous secretion of estrogen which causes persistent vaginal corni-
fication. A temporary state resembling these prolonged estrous condi-
tions can be produced in the rat by neural blocking agents presented
before the time of onset of the critical period for LH release (2-4 PM).
If such injections are repeated for several days, the animals appear to
continue secreting estrogen until atresia occurs (Everett, 1961).

Light induced persistent estrus appears to resemble the proestrous
stage of the estrous cycle, except for the cornified vaginal cytology.
With a strong stimulus such as mating, ovulation occurs in these animals.
Therefore it was of interest to determine whether light induced per-
sistent estrous rats showed daily patterns of LH, FSH, and Prl release
similar to those seen in proestrous.

B. Methods and Materials

 

Mature Sprague-Dawley female rats weighing 225-275 grams were

either housed under 14:10 1ight:dark conditions or under constant light

70

71

as described in the Methods section. Rats kept in the light:dark regimen
(LD) were selected on the day of proestrous of a normal 4-day estrous
cycle. Animals kept in constant light (LL) for at least 5 weeks and
showing 10 consecutive days of vaginal cornification were considered to
be in persistent estrus, and used for this experiment. Blood samples
were collected by cardiac puncture under light ether anesthesia in the
morning (0900—1000 h) and afternoon (1600-1700 h). Serum levels of LH,
FSH, and Prl were determined by radioimmunoassay as described in the
Materials and Methods section.
C. Results

Morning levels of serum LH were not significantly different in
LL and LD rats (22:10 ng/ml vs. 20:8 ng/ml), respectively (Figure 2).
A significant increase in LH was seen in the LD proestrous rat in the
afternoon but not in the LL rat (508:115 ng/ml vs. 24:6 ng/ml (Figure 2)
(P<0.05). These results indicate that the times measured, no preovula-
tory LH surge was seen in rats exposed to constant light.

Morning levels of serum FSH were slightly higher in LL rats than
in LD rats (233:16 ng/ml vs. 196:8 ng/ml) (Figure 2) (P<0.05). However,
both groups of animals showed an afternoon rise in FSH which was of
equal magnitude (510:26 ng/ml vs. 526:54 ng/ml). These results demon-
strate that unlike LH, constant light apparently does not suppress the
ability of FSH serum levels to surge in the afternoon. Prl morning
serum levels were elevated in LL rats as compared to LD rats (38:8
ng/ml vs. 7:4 ng/ml) (Figure 2) (P<0.05). In contrast to the afternoon
surge of Prl seen in the LD rats (317:124 ng/ml), LL rats showed no
significant difference between AM and PM serum levels. These results

suggest that although LL may induce elevated basal levels of serum Prl

72

 

 

 

 

ll
to
an an m
A M
E z
2 an / 5m: ,.
o / a
O , a
s ‘°° ‘°° a
\ -
‘g 3“) 3
2
3 /’ '00 3
.0 ,/ v
‘wo um
AM PM PM
TIMI
Figure 2. The Effect of Constant Light on Serum LH and FSH

Levels in the Morning (AM) and Afternoon (PM).

Serum LH and FSH concentrations at 0900-1000 h (AM)
and 1600-1700 h (PM) were compared in proestrous rats
kept under 14L:10D conditions (LD) (open bars) and
light induced persistent estrous rats (LL) (striped
bars). Each bar represents the mean determination

of 6-8 rats. Vertical lines indicate :1 SEM.

73

9" ll
II to

 

 

400
I
g 300
3
2 200
E
100 L
.v’v
40
20
AM PM
tun!
Figure 3. The Effect of Constant Light on Serum Prl Levels

in the Morning (AM) and the Afternoon (PM).

Serum Prl concentrations at 0900-1000 h (AM) and
1600-1700 h (PM) were compared in proestrous rats kept
under 14L:10D conditions (LD) (open bars) and light
induced persistent estrous rats (LL) (striped bars).
Each bar represents the mean determination of 6-8
rats. Vertical lines indicate :1 SEM.

74
as compared to LD proestrous rats, the afternoon Prl surge was not seen
at this sampling time. It is however, possible that a Prl surge may
have occurred at some other time.

D. Discussion

 

From these data, it might be suggested that constant light
exposure may have a differential effect on LH and FSH release. The
characteristic proestrus afternoon surge of LH was not seen in the LL rat.
This is what might be expected based on the previous observations of
Daane and Parlow (1971) who reported a greatly reduced incidence of the
normal proestrous LH surge even during the first vaginal cycle following
exposure to constant light. Serum FSH levels have been reported to be
elevated in LL rats (Fink, 1975), but the present data are believed to
be the first observations of a proestrous type afternoon "surge" of FSH
in LL rats. Since all of the LL rats showed a rise in FSH on the same
day, it is likely that this may be a daily occurrence in light induced
persistent estrus. Perhaps this daily pulse of FSH release participates
in maintaining a constant secretion of estrogen. This effect could in
turn disrupt gonadal steroid positive feedback of LH release if the
control mechanism for that release was rate dependent.

The differential effect of LL on LH and FSH release is also in-
teresting from the standpoint that it supports the theory for separate
releasing factors for LH and FSH rather than a common one. This obser-
vation might also suggest that constant light may be selectively
affecting hypothalamic neuronal centers associated with LH and Prl
positive feedback. Possibly these separate gonadotropin control systems

are desynchronized and unmasked by the LL treatment. In support of the

75
differential effect which LL has on gonadotropin release are the obser—
vations of Piacsek §£_al, (1966). They observed a release of FSH from
pituitary transplants in hypophysectomized rats when placed in constant
light. These investigators also found that serum levels of FSHRH (as
measured by bioassay) were elevated in those rats.

Anterior hypothalamic lesions can accelerate the onset of the breeding
season in ferrets (Donovan g£_§l,, 1956). It is possible that the lesions
produce estrus by the destruction of a neural mechanism which, during
sexual quiescence, would restrain gonadotropin secretion. Stimuli such
as constant light, might have this same effect by depressing this neural
mechanism, and a discharge of gonadotropin (primarily FSH) in an amount
sufficient to stimulate the gonads would follow.

According to Legan and Karsch (1975), a neural signal for the LH
surge is emitted each day throughout the estrous cycle of the rat, and
prolonged maintenance of elevated circulating estradiol is essential
for the expression of these signals. Perhaps a rate sensor exists for
the rapid increase in circulating estradiol which can be remembered for
at least several days afterwards.

Unlike the proestrous rat, no afternoon Prl surge was seen in the
LL rat. It is not clear what role Prl plays in maintenance of the
estrous cycle. Therefore, the significance of the absence of a Prl
surge in the LL rat is difficult to determine. It cannot be concluded
at this point whether the lack of a Prl surge is the result or one of
the contributing factors in the light induced persistent estrous
syndrome.

Although light induced persistent estrus is somewhat sbmilar to

proestrus, the afternoon surges of LH and Prl are not seen in this

76
condition. In contrast, a surge of FSH is seen in the afternoon of LL
rats. Although the etiology of light induced persistent estrus is
still unclear, the changes in gonadotropin release observed in this
experiment probably reflect effects rather than causes of this condition.

II. Effect of Constant Light on the Sensitivity of the Pituitary to
Exogenous GnRH In Vivo in the Mature Female Rat; Single and Multiple

 

 

Injections of §ynthetic GnRH

 

A. Objective

In normal cycling mature female rats the reSponsiveness of the
anterior pituitary to ex0genous or endogenous luteinizing hormone re—
leasing hormone (LHRH) changes with the various stages of the estrous
cycle (Aiyer g£_§l,, 1973, 1974; Cooper g£_al,, 1973; Fink and Aiyer,
1974; Gordon and Reichlin, 1974). The timing and magnitude of the in-
crease in pituitary responsiveness indicates that it plays a major role
in the development of the LH surge. Conceivably, constant exposure of
rats to light may also lead to an increase in pituitary responsiveness
to LHRH which would facilitate the rapid reflex release of LH after
mating (Brown-Grant 25 31., 1973) and the postulated increase in the
secretion of FSH (Critchlow, 1963) in this type of animal. Fink (1975)
has shown that the pituitary of the constant light exposed rat responded
to synthetic doses of LHRH similarly to regular cycling rats on the
morning of proestrus. The purpose of this study was to further in-
vestigate the possibility that increased pituitary responsiveness to
LHRH occurs in constant light, especially the pituitary response to

single and multiple priming doses of synthetic LHRH.

77

B. Materials and Methods

 

Experiments I and II

 

Female Sprague-Dawley rats weighing 225-275 g were exposed to
constant light for about five weeks. Only rats showing at least 10 con-
secutive days of vaginal estrus were considered to be in persistent
estrus and used in this study. Control animals of this study were female
rats of the same age, strain and weight as above and selected on their
ability to show a regular 4-day estrous cycle under 14L:10D lighting con-
ditions. The control group was divided into proestrus, estrus, and
diestrus (I and II). All experimental procedures were done between 0900
and 1000 h.

Synthetic gonadotropin releasing hormone (GnRH) (Abbot, Chicago, IL)
was dissolved in 0.89 percent NaCl in concentrations of 450 ng or 50 ng
GnRH/0.1 ml. After pretreatment blood samples were obtained by cardiac
puncture under light ether anesthesia, the animals were given a single
injection of 450 ng GnRH/100 g body weight (Experiment I) or six injec-
tions of 50 ng GnRH/100 g body weight every 30 minutes for a period of
two and one-half hours (Experiment II). All injections were administered
subcutaneously in a volume of 0.1 m1/100 g body weight.

In Experiment I, blood samples were taken at 45 minutes prior to a
single injection of 450 ng GnRH/100 g body weight and at 15 and 75
minutes thereafter.

In Experiment II, serial blood samples were taken at -15, +30, 60,
120, and 180 minutes following the initial injection of 50 ng GnRH/100
g body weight. An additional proestrous group was added and was bled
and injected according to the same schedule as above between 1500 and

1700 h.

78
Serum LH and FSH were measured by standard double-antibody radio-
immunoassay procedures using NIAMDD kits. Results are expressed as
ng/ml serum in terms of the reference standards, NIAMDD rat LH-RP-l and
NIAMDD rat FSH-RP-l.
C. Results

Effect of a Single GnRH Injection on Serum LH in LL and LD Rats

Pre-injection serum LH levels were not significantly different
among any of the groups in this experiment. The LH response to a single
injection of 450 ng GnRH/100 g body weight is shown in Figure 4. Serum
LH rose in the LL rats from 21:5 ng/ml to 442:19 ng/ml at 75 minutes
after injection of GnRH. Proestrous and estrous rats showed an LH
response of similar but lesser magnitude at 75 minutes (19:4 ng/ml to
234:23 ng/ml and 18:2 ng/ml to 240:45 ng/ml, respectively). In contrast,
the diestrous group responded to the GnRH injections with a serum LH
increase from 19:1 ng/ml to 51:12 ng/ml at 75 minutes. There was no
significant difference in serum LH concentration changes among control
groups between 15 and 75 minutes after GnRH injection. Serum LH levels
of LL rats, however, appeared to continue rising even at 75 minutes
(442:19 ng/ml) and was significantly greater than serum LH levels of
any of the control groups (P<0.05) at that time. This would indicate a
greater pituitary LH response to GnRH in LL rats.

Effect of Multiple GnRH Injections on Serum LH in LL and LD
Rats

 

The effects of a series of six injections of 50 ng GnRH/100 g
body weight are shown in Figure 6. Serum LH rose from 19:3 ng/ml to
1226:206 ng/ml in LL rats and from 19:4 ng/ml to 1128:194 ng/ml in pro-

estrous (PM) rats in 180 minutes following the initial GnRH injection.

79

ll.
Fr.
In
Dion

2

8

b

O

O

i am:

\

I

5

3 200

100

 

Figure 4. The Effect of Constant Light on Pituitary LH Response

to a Single Synthetic GnRH Injection In_Vivo.
Synthetic GnRH (450 ng/kg body weight) was injected
s.c. into constant light (LL), and in proestrous (Pro),
estrous (Est) and diestrous (Diest) rats (14L:10D).
Each bar represents the mean determination of 6-8 rats.
Vertical lines indicate :1 SEM.

80

ll.
he
In
Nos.

500

400

I!" (In. (ml usual)

 

 

 

 

 

- u . 0 1'
MINI]!!!
On!"

The Effect of Constant Light on Pituitary FSH Response

Figure 5.
to a Single Synthetic GnRH Injection Ig_Vivo.

See Figure 4 for explanation.

Figure 6.

‘ l" (on/ml serum)

81

Pituitary Response To Multiple
Gull-l Inleetiem

I
I Preeu.
/’ W"
[I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
'I
P! O.
I 0.... ...... .n...
' ...'. '0‘.
)‘.e0'”.
/ "M 0“...
I
.. .-
4: 09.30:». ouoo one
. | . ‘
Onltt Minutes

The Effect of Constant Light on Pituitary LH Response
to Multiple Synthetic GnRH Injections 12_Vivo.

Synthetic GnRH (50 ng/kg body weight x 6) was injected
s.c. into constant light (CL) rats and in proestrous
(AM), (PM), estrous, and diestrous rats kept under
14L:10D conditions. Blood samples were collected at
-15, +30, 60, 120, and 180 minutes following the initial
injection of a series of six GnRH injections given every
30 minutes (arrows). Each point represents the mean
determination of 6-8 rats. Vertical lines indicate

:1 5m.

82

Pituitary Response To Multiple
GnRH lnleetipns

 

 

 

? .
a 900
3
"i no
\
e
5.
5 500

300

‘00

.15 0 ‘QQO ‘ 60 t ‘ 120‘ t m
can Minutes
Figure 7. The Effect of Constant Light on Pituitary FSH ReSponse

to Multiple Synthetic GnRH Injections ln_Vivo.

See Figure 6 for explanation.

83
The LH responses of these two groups to the GnRH injections were not
significantly different from one another. Pretreatment serum LH levels
of proestrous (AM) (16:2 ng/ml), estrous (14:3 ng/ml), and diestrous
(12:3 ng/ml) rats rose to 413:185 ng/ml, 130:7 ng/ml, and 152:6 ng/ml,
respectively, in 180 minutes following GnRH injections. This would
suggest that the priming effect of GnRH is greater in LL rats and pro-
estrous (PM) rats than in proestrous (AM), estrous and diestrous rats
in terms of pituitary LH response.

Effect of a Single GnRH Injection on Serum FSH in LL and LD
Rats

Basal levels of serum FSH in the control groups of animals
were not significantly different from one another. Basal levels of LL
rat serum FSH was significantly greater than those of any of the control
groups (Figure 5). There was no difference in the magnitude of increase
in serum FSH in any of the groups 15 minutes following a single injec-
tion of GnRH. However, 75 minutes after the GnRH injection LL rats had
significantly higher (P<0.05) serum FSH levels (561:91 ng/ml) than pro-
estrous (428:22 ng/ml), estrous (456:31 ng/ml) or diestrous (412:43
ng/ml) rats.

Effect of Multiple GnRH Injections on Serum FSH in LL and LD
Rats

 

Pituitary FSH response to six injections of 50 ng GnRH/100 g
body weight in LL and LD rats is shown in Figure 7. Basal levels of
serum FSH in LL rats was 310:20 ng/ml which was not different from pro-
estrous (PM) (29l:62 ng/ml), proestrous (AM) (288:88 ng/ml) but greater
than estrous (225:18 ng/ml) or diestrous (227:7 ng/ml) rats (P<0.05).

At 180 minutes after the start of the GnRH injection series, serum

FSH levels rose to 770:72 ng/ml in LL rats. This was not different from

84

proestrous (PM) rats (652:48 ng/ml) but significantly greater than pro-
estrous (AM) (581:33 ng/ml), estrous (404:41 ng/ml) or diestrous
(345:28 ng/ml) rats.

D. Discussion

 

The LL rats showed a greater pituitary LH and FSH response to
a single injection of 450 ng GnRH/100 g body weight than any of the LD
control groups. Furthermore, there appeared to be a difference in the
time course of the response of pituitary FSH and LH to single GnRH
injection between LL and LD control groups. While LD control rats
showed their maximum serum levels of FSH and LH within 15 minutes
following GnRH injection, the highest serum levels of FSH and LH did not
appear until 75 minutes following the GnRH injection in LL rats. This
difference may suggest a greater pituitary reserve of LH and FSH in
the LL rat but not necessarily a greater rate of release of these gonado-
tropins. In contrast, Fink (1975) found that the increments in plasma
LH and FSH of LL rats 60 minutes following a 50 ng dose of synthetic
GnRH were similar to those of proestrous animals. The difference in
results of these two experiments could be due to differences in the
doses of GnRH used or to route of administration or blood sampling
times.

In animals exposed to constant light, mating causes a prompt in-
crease in serum LH which results in ovulation (Brown-Grant gt $1.,
1973; Smith and Davidson, 1974). However, this particular sensitivity
of the LL rat to ovulation-inducing stimuli is not the result of a
supersensitive pituitary to LHRH (Smith and Davidson, 1974). In order

to elicit an LH response to GnRH in LL rats which was comparable to

85
that response seen in the LD rat during the proestrous preovulatory
period (PM), Fink (1975) found that it was necessary to inject 4-5
times the dose of GnRH. Therefore, Fink suggested that in contrast to
the normal animal, in which only a small fraction of the readily re—
leasable pool of LHRH is discharged during the Spontaneous preovulatory
surge, the mating reflex surge of LH in LL rats involves the release of
most of the readily releasable pool of the hypothalamic hormone.
According to Aiyer E£.§l° (1974), the magnitude of the LH response is
related in a positive manner to the secretory activity of the pituitary
before administration of GnRH. This relationship may be due to a priming
effect of GnRH on the pituitary.

The priming effect of GnRH on pituitary LH and FSH release was also
compared between LL and LD control rats in this study. LL rat pituitary
LH and FSH release was not significantly different from that of pro-
estrous (PM) rats in response to multiple priming doses of GnRH but
significantly greater than proestrous (AM), estrous, or diestrous rats.
Therefore, the magnitude of the LH and FSH response to priming doses of
GnRH in LL rats is comparable if not greater than regular cycling
animals in proestrus.

Fink (1975) suggested that the increased responsiveness of the
anterior pituitary in rats exposed to constant light may be related to
the levels of circulating estradiol which are higher than at estrus
but lower than at proestrus in rats with 4-day cycles (Naftolin st 31.,
1972).

The results of this study would argue against the possibility that

in the constant light exposed rat, the failure of spontaneous

86
preovulatory LH release and corresponding ovulation is due to a de-
creased pituitary sensitivity to LHRH.
III. Effect of Constant Light on the Content of LHRH in the Anterior

Hypothalamic Area (AHA) and Medial Basal Hypothalamus (MBH) of the
Mature Female Rat on Morning and Afternoon Time Periods

A. Objective
Exposure of the pituitary to increased amounts of hypothalamic

LH releasing hormone (LHRH) is believed to be a direct stimulus for the
preovulatory LH release in the normal cycling female rat. Unfortunately,
LHRH secretion cannot be measured without severely stressful hypophysial
portal vessel cannulation procedures. An alternative approach to in-
vestigating the role of LHRH is to measure its content in the hypothala-
mus. A depletion of LHRH content in the hypothalamus occurring prior to
an event such as the preovulatory LH surge may suggest release of hypo-
thalamic LHRH into the portal circulation to stimulate the LH surge.

Since light induced persistent estrous rats fail to show any LH
surge to stimulate ovulation, it was of interest to test the hypothesis
that these animals had either a deficiency of LHRH in the hypothalamus,
or that the hypothalamic LHRH was not being released into the portal
vessels to stimulate pituitary LH release.

B. Methods and Materials

 

Mature female Sprague—Dawley rats weighing 225-275 g were kept
under constant light for 5 weeks. Those animals showing at least 10
consecutive days of vaginal estrus were considered to be in persistent
estrus and used in this experiment. A control group of rats was kept
under 14L:10D light conditions and selected on the day of proestrus of

a regular 4-day cycle.

87

Rats were killed by decapitation in the morning (0900-1000 h) or
afternoon (1600-1700 h). Brains were removed from the cranium imme-
diately after killing and dissected according to the procedure outlined
in the general Materials and Methods section. Hypothalamic LHRH was
measured by the double antibody RIA method of Nett £5 21, (1973).

C. Results

Anterior hypothalamic area (AHA) concentrations of LHRH in LL
rats was higher than LD proestrous rats in the AM (817:96 pg/hypothala-
mus vs. 494:24 pg/hypothalamus) and in the PM (551:68 pg/hypothalamus
vs. 244:20 pg/hypothalamus) (Figure 8). Medial basal hypothalamus (MBH)
concentrations of LHRH were also higher in the LL rat than LD control
at both AM (6.4:0.4 ng/hypothalamus vs. 4.8:0.2 ng/hypothalamus) and
PM (6.5:0.7 ng/hypothalamus vs. 3.8:O.2 ng/hypothalamus) time periods
(Figure 8).

These results suggest that no deficiency of LHRH exists in the LL
rats as compared to the LD proestrous rat. In fact an apparent surplus
of LHRH is seen in both areas of the hypothalamus examined in the LL
rat .

AHA LHRH concentrations were significantly smaller in the PM than
AM (244:20 pg/hypothalamus vs. 494:24 pg/hypothalamus) (P<0.05) in the
LD proestrous rats. This same pattern was seen in the AHA of the LL
rat. Afternoon (PM) concentrations of LHRH were smaller than AM con-
centrations (551:68 pg/hypothalamus vs. 817:96 pg/hypothalamus) (P<0.05)
in LL rats (Figure 8). These results suggest that AHA LHRH concentra-
tions change with time in both types of animals. This change in

content with time may imply movement of LHRH from one area of the

88

ll
l0

 

as» AHA
O
8
3 E
3
2 z
i z
2 3.
\ 3’
U C
‘ i’
3
3 5
I
B
a
Figure 8. Effect of Constant Light on LHRH (GnRH) Content of

the Preoptic-Anterior Hypothalamic Area (AHA) and the
Medial Basal Hypothalamus (MBH) in the Morning (AM)
and Afternoon (PM).

GnRH content of both hypothalamic areas was measured in
constant light (LL) rats and proestrous rats kept under
14L:10D conditions (LD) in the morning (AM) and after-
noon (PM). Each bar represents the mean determination
of 6—8 rats. Vertical lines indicate :1 SEM.

89

hypothalamus to another during the time course of hypothalamic events
associate with the estrous cycle.

Medial basal hypothalamic LHRH content was smaller in the PM than
AM (3.8:0.2 ng/hypothalamus vs. 4.8:0.2 ng/hypothalamus (P<0.05) in the
LD controls (Figure 8). This pattern was not seen in the MBH of LL
rats comparing PM to AM levels of hypothalamic LHRH (6.5:0.7 ng/hypo—
thalamus vs. 6.4:0.4 ng/hypothalamus). These results suggest that while
LHRH is removed from the MBH by the afternoon time period (i.e., pre-
sumably partially released to the portal vessels) in the LD proestrous
rat, no such release of LHRH was seen in the LL rat.

D. Discussion

 

The results of this experiment suggest that LHRH is present
in the hypothalamus of LL rats in quantities which exceed those of the
LD proestrous rat. However, the data also infer that unlike the LD
rat, the LL rat showed no apparent "release" of LHRH from the MBH. It
should be noted that since LHRH was measured at only one time in the
PM in this experiment, a different time course of LHRH release in the
LL rat cannot be ruled out. It is unlikely that this occurred since
ovulation did not follow.

As an alternative to increased LHRH release, the preovulatory
surge of LH may result from an increase in pituitary sensitivity to
LHRH. This hypothesis was tested and discussed in Experimental section
II of this thesis. To summarize, it was found that the pituitary of
the LL rat was equally or more sensitive than the pituitary of the LD
proestrous rat to priming doses of GnRH in zigg. Therefore, endogenous
hypothalamic LHRH content of LL rats was examined in this experiment

and compared to that of the proestrous rat.

90

One feature of the rat exposed to constant light is that is is
quite sensitive to ovulation-inducing stimuli. Smith and Davidson (1973)
found that 100 percent ovulation occurred following stressful stimuli
in these rats. They observed a decline in LHRH activity of the basal
hypothalamus of LL rats which reached a low point 20 minutes after the
onset of mating, followed by a return toward the initial level, suggesting
rapid release of stored LHRH followed by resynthesis. These observations
would support the hypothesis that no deficiency of hypothalamic LHRH
exists in the LL rat but that its "spontaneous" (but not "reflex")
release may be impaired.

Another characteristic of constant light exposure is the advancement
of the average age of vaginal opening and the first appearance of large
follicles in immature rats (Steger 23 31., 1975). These effects may be
associated with alteration in follicle stimulating hormone releasing
hromone (FSHRH) levels in the immature LL rat as described by Negro-
Vilar gt_§;, (1968). These workers found elevated levels of FSHRH
activity in plasma of hypophysectomized rats exposed to constant light
which were greater than that of LD hypophysectomized rats. Increased
FSHRH activity could presumably release FSH which could stimulate
follicular growth and estrogen production by ovaries to result in
advancement of vaginal openings, but ovulation does not occur. This
latter effect may be due to an inability of the LL rat to spontaneously
release LHRH. Since FSH serum levels are elevated in the adult LL rat
while no change in LH serum levels is seen, this differential release
of gonadotropin releasing factors may be occurring in the adult LL rat

as well.

91

This experiment has presented at least some indirect evidence which
suggests that LHRH is present but not released from the hypothalamus in
light induced persistent estrus.

IV. Effect of Constant Light on Hypothalamic Sensitivity to Positive

and Negative Feedback by Gonadal Steroids: LH and FSH Response to
Estradiol Benzoate (EB) and Prqgesterone (P) Priming

A. Objective

There is abundant evidence that ovarian steroids can facilitate

 

or inhibit luteinizing hormone (LH) secretion and ovulation depending
on the dose administered and the physiological conditions at the time
of the hormone treatment (Everett ggngl., 1969; Brown-Grant, 1974).
The preovulatory release of gonadotropin in cycling rats is also in-
fluenced by a neural mechanism which is cued by the light-dark cycle
(Everett ggflal., 1949; Critchlow, 1963). Phase shifts in the onset of
the light period cause a corresponding temporal shift in the activation
of the control mechanism and consequently of pituitary LH discharge.
In ovariectomized rats, the timing of estrogen—induced LH release can
also be altered by advancing the onset of the light period (Columbo g;
.31., 1974). Thus, the environmental light cycle acts as an external
signal for synchronizing the pituitary discharge of LH in both cycling
and estrogen-primed ovariectomized animals.

The preovulatory rise in estrogen titers is known to be an impor-
tant factor in spontaneous LH release and ovulation (Brown-Grant, 1974;
Schwartz, 1969). A number of factors could affect estrogen secretion
rate and LH-release threshold, although no specific data are available
to distinguish between these two sites of action. The effects of

photoperiod on cycle length might be due to changes in the LH-release

92

threshold (Hoffmann, 1975). In the extreme conditions of constant
light, the LH release threshold may be raised and estrogen secretion
rate slowed.

It was the objective of these experiments to compare the thresholds
for positive and negative feedback of estrogen and progesterone on LH
and FSH release between ovariectomized rats exposed to constant light
and ovariectomized rats exposed to a 14L:10D light cycle.

B. Methods and Materials

 

Female Sprague-Dawley rats weighing 225-275 g were housed
under constant illumination for a period of about five weeks. A second
group of Sprague-Dawley females was kept in a 14L:10D lighting regimen.
Those animals showing persistent estrus in the constant illumination or
those control animals showing regular cycles in the 14L:10D regimen
were then ovariectomized. Two to three weeks later, blood samples were
taken by cardiac puncture under light ether anesthesia. Serum was
separated by centrifugation and stored at —20°C. Serum LH and FSH was
measured by RIA as outlined in the general Materials and Methods section.

Estrpgen and Prpgesterone Priming

Experiment I — Twenty ug of estradiol benzoate (BB) in 0.2 ml

 

corn oil was administered S.C. to each group of rats. Seventy-two hours
later a second injection of 20 ug EB was given. Thirty hours after the
second injection, a blood sample was taken by cardiac puncture under
light ether anesthesia.

Experiment II - Twenty ug of EB in 0.2 ml corn oil was admini—

 

stered 8.0. to each group of rats. This was followed seventy-two

hours later by 5 mg progesterone (P) administered S.C. in 0.2 ml corn

93
oil. Six hours after the P injection, a blood sample was taken by
cardiac puncture under light ether anesthesia.
C. Results

Experiment I

 

The effects of castration and EB positive and negative feed-
back on serum levels of LH and FSH in LL and LD rats is seen in Figure
9. Post-castration levels of LH were not different between LL and LD
rats (560:161 ng/ml and 419:92 ng/ml, respectively). Seventy-two hours
following 20 ug EB, serum LH was depressed to a lower level in LB rats
(86:17 ng/ml) than LL rats (352:170 ng/ml (P<0.05). In response to a
second injection of EB, LD rats showed a small but significant increase
in serum LH (273:167 ng/ml) while serum LH levels continued to fall in
LL rats (104:39 ng/ml) at this time.

No differences were seen in serum FSH levels between LL and LD rats
in response to castration nor after negative or positive EB stimuli.

Experiment II

 

The effects of castration, EB negative and P positive feedback
on serum levels of LH and FSH in LL and LD rats is seen in Figures 10
and 11. Serum LH levels of LL and LD rats did not differ prior to nor
at 1 or 12 days postcastration (Figure 10). Although LL rats showed a
greater EB negative feedback on serum LH than LD rats, comparing post-
castrate and post-EB serum LH levels, both groups of rats responded
similarly to progesterone treatment with an elevation of serum LH
(Figure 10). This would indicate that progesterone can overcome any
difficulty in positive feedback of serum LH release under constant

light conditions.

Figure 9.

Hi (nglul)

.00

  

94

2000

u ’5
Lo 5;

I 1200

(wise) as:

 

 

Cestrete‘ 72hr ‘ 30hr ‘..".n° 72.» ‘ 30'"
'I P. P. I.

The Effect of Constant Light on Estradiol Benzoate (EB)
Induced Negative and Positive Feedback Effects on Hypo-
thalamic Control of Pituitary LH and FSH Release.

Constant light rats (LL) and rats kept under 14L:10D con-
ditions (LD) were ovariectomdzed and injected with EB

(20 ug) s.c. at 72 hr intervals after which gonadotropin
response to negative (72 hr) and positive (30 hr) feed-
back of EB was measured. Each bar represents the mean

determination of 6-8 rats. Vertical lines indicate
:1 SEM. ‘

Figure 10.

I. It (up, all serum)

95

1000 "°

“00

12

000

200

       

1 s 12 a -. 24 In f s In
Pf. - 1 ;
sestrete pestCesttete II P

Effect of Constant Light in Progesterone (P) Induced
Positive Feedback on Hypothalamic Control of Pituitary
LH Release.

Constant light (LL) rats and rats kept under 14L:10D
conditions (LD) were ovariectomized and injected with
estradiol benzoate (EB) (20 ug) followed 24 hr later
by progesterone (P) (5 mg) s.c. Gonadotropin response
to P positive feedback was measured 6 hr following P
injection. Each bar represents the mean determination
of 6-8 rats. Vertical lines indicate :1 SEM.

ISM ("ml serum)

Figure 11.

96

ll
LD

 
    

\ x,
\ \\\\\
~ \\\\ \ \ ‘3 .
\ \\\\\:\\§\\\k\t\\\\\ \ \ '\ \ \ ~

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ix \
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§&<\.
i§fi\<§\g\v\qlvk g
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Pre- Id d 7 24hr ? 6hr
sestrete Pesteestrete '1. 1'

Effect of Constant Light in Progesterone (P) Induced
Positive Feedback on Hypothalamic Control of Pituitary
FSH Release.

See Figure 10 for explanation.

97

Basal serum FSH levels were not different between LL and LD rats
(Figure 11). However, 1 day following castration LD rats had higher
serum FSH levels than LL rats (755:44 ng/ml vs. 568:31 ng/ml). Similar-
ly at 12 days post-castration serum FSH levels in LD rats were still
higher than those of LL rats (2912:39 ng/ml vs. 2256:93 ng/ml). Six
hours following an injection of P (5 mg) serum FSH levels of LD rats
rose to a higher level than in LL rats (4637:872 ng/ml vs. 2727:226
ng/ml). This would suggest that, at six hours after progesterone
injection serum FSH levels of LL rats were not as sensitive to positive
feedback by progesterone.

D. Discussion

 

Since spontaneous ovulation does not occur in light-induced
persistent estrus, it was of interest to test the response of gonadotro-
pin release in the LL rat to negative and positive feedback effects of
gonadal steroids.

The ability of EB to exert a negative feedback effect on the post-
castration serum LH rise was demonstrated in LB rats but not LL rats in
exerpiment 1 (Figure 9) 72 hr after injection. In contrast, the results
of experiment 2 show the opposite LH response but at 24 hr after EB
injection (Figure 10). The discrepancy in LH response between these
two experiments is probably due largely to a difference in sampling
times following EB injection. This also suggests that in the LL rat,
there may be a different time course for steroid negative feedback on LH
release. Alternatively, if LH responses between LL and LD rats were
compared only at 72 hr after EB, the data may suggest that the sensi-
tivity of the LH control system to the negative feedback effects of

gonadal steroids was decreased in constant light. This latter

98
alternative is in agreement with the findings of Hoffmann and Cullin
(1977) demonstrating a decreased negative feedback sensitivity of the
LH control system to estrogen negative feedback with increased photo—
period length. Unlike LH, the FSH response to negative feedback by EB
was not different between LL and LD rats.

Estrogen not only has negative feedback effects on LH release, but
also can induce the release of a surge of LH comparable to the ovulatory
surge in rhesus monkeys (Karsch g£_al., 1973; Legan g£_§l,, 1975).
Differences between the LH response to the positive feedback effects of
estrogen were also seen between LL and LD rats. In contrast to LD
rats, LL rats failed to show a serum LH increase following a second EB
injection in experiment 1 (Figure 9). Since serum was collected at
only one time period following the second EB injection, it cannot be
concluded whether or not the sensitivity of LH release control to
positive feedback by EB is decreased in the LL rat or whether the time
course of LH response was different in the LL rats. However, in a
related study, Mennin and Gorski (1975) found no elevation of serum LH
following a second EB injection in ovariectomized light-induced persis-
tent estrous rats at time periods ranging from six to fifty-six hours
following the EB injection.

Mann 2; a1. (1976) have shown that in the ovariectomized rats in
a 14L:10D light cycle: a) EB produces an LH rise in the absence of any
circulating progesterone (P), b) and P potentiates and advances the
serum LH surge in response to EB. These findings are in agreement
with our observations in experiment 2. In addition, Mann fig 2;. (1976)
have shown that rats maintained in constant light were less sensitive

to the positive feedback actions of EB and P than LD rats. In

99H

experiment 1, the LL rats failure to respond to the positive feedback
of EB is in agreement with the results of Mann.££ El: (1976). However,
in experiment 2, in contrast to the findings of Mann gg'gl. (1976), a
decreased sensitivity to the positive feedback effect of P in LH was
not seen. A possible explanation for this discrepancy may be the
difference in doses of P used in the experiments of Mann ggmgl. (1976)
and experiment 2. Since almost twice as much P was used in our experi-
ment 2 as compared to theirs, a higher serum level of P may have been
able to overcome a decreased sensitivity of the LL rat to the positive
feedback effects of P on LH.

It is interesting to note that in the studies of Mann 35-21: (1976),
the degree to which sensitivity of positive feedback of EB and P on LH
was reduced was dependent on whether or not the animals had adrenal
glands. Rats that were both ovariectomized and adrenalectomized showed
an enhanced sensitivity to EB and P in both LL and LD rats. No expla-
nation was given for this finding.

The results of these experiments would suggest that LL induces an
alteration in the sensitivity of the control system for LH release to
negative and positive feedback by gonadal steroids. This may contri-
bute to a lack of spontaneous ovulation in the light induced persistent
estrous rat.

V. Effect of Constant Light on Morning and Afternoon Plasma Levels of
Estradiol, Progesterone and Testosterone in the Mature Female Rat

A. Objective

The sequence of changes in plasma gonadal steroids which occurs

in the normal cycling female rat are believed to contribute to positive

100
and negative feedback effects on gonadotropin secretion (Everett, 1964;
Kalra and Kalra, 1974). They participate in the regulation of hormonal
events occurring during the estrous cycle. The persistent estrus
resulting from constant light exposure is associated with an absence of
the preovulatory surge.

It was of interest to measure plasma estradiol, progesterone and
testosterone levels in light induced persistent estrous rats. It has
been suggested that the receptivity of the constant light exposed rat
to a mating stimulus and its effectiveness in inducing ovulation may be
due to suprabasal but constant secretion of estrogen maintaining the
estrous vaginal cytology and priming the pituitary to respond to an LHRH
stimulus (Smith and Davidson, 1974).

B. Methods and Materials

 

Mature Sprague-Dawley female rats were maintained under 14L:10D
or constant light (LL) conditions. LD rats showing a regular 4-day
estrous cycle were selected for this experiment on the day of proestrous
of the estrous cycle. LL rats which showed ten consecutive days of
vaginal estrus were also selected for this experiment. Blood samples
were collected by cardiac puncture under light ether anesthesia in the
morning (0900-1000 h) and afternoon (1600-1700 h). Plasma steroids
were measured according to the procedure described in Appendix D.

C. Results

Plasma estradiol levels of LL rats were not significantly
different from LD proestrous rats in the AM (34:4 Pg/ml vs. 28:8 pg/ml).
However, while estradiol levels in proestrous rats rose to 44:7 pg/ml in
the afternoon, LL rats showed no significant change in estradiol levels

at this time (Figure 12).

101

Plasma progesterone levels were significantly lower in LL rats than
proestrous rats at morning (12:4 ng/ml vs. 26:8 ng/ml) (P<0.05) and
afternoon (16:4 ng/ml vs. 34:9 ng/ml) (P<0.05) time periods (Figure 12).

Plasma testosterone levels were higher in LL rats than LD proestrous
rats in the morning (203:38 pg/ml vs. 84:12 pg/ml) (P<0.05), but this
relationship was reversed in the afternoon when LL plasma testosterone
levels fell to 130:9 pg/ml while LD proestrous levels rose to 324:41
pg/ml (Figure 13).

D. Discussion

 

Data on plasma estradiol levels in this experiment show that at
those time periods when samples were collected, LL rats had plasma
estradiol levels which were neither much higher nor lower than the range-
of estradiol concentrations found in the normal cycling rats. This
observations is supported by the findings of Takahashi SE 2;. (1977)
where ovarian vein plasma levels of estradiol were of an intermediate
value between those of diestrous night and proestrous morning in normal
cycling rats.

The interpretation given by various investigators concerning the
basis for persistent estrus that develops after exposure to constant
light centers around the concept of a persistent mild elevation in
gonadotropin secretion (FSH) (Daane and Parlow, 1971; Mann £5 31.,

1973, 1974) that drives a slightly elevated estrogen secretion (Nafto-
lin ggflgl., 1972) hovering at a level intermediate between the peaks and
troughs of the normal cycle. There is also a reduction in estrogen
binding capacity at various estrogen target tissues in long-term 1ight-
sterilized rats (Allei-Donhoffer g£_§l,, 1974) which may be related to

a failure of the normal feedback regulation of ovulation.

102

ll.
LD

   

    

3
iso w§
~ 3
‘ =
a” so:

I
3 2‘
3 so 20 r
a 1

.s
0

AM PM . AM

.
"Ml
Figure 12. Effect of Constant Light on Plasma Estradiol and
Progesterone Levels in the Morning (AM) and After-

noon (PM).

Morning and afternoon levels of plasma estradiol and
progesterone were measured in constant light (LL) rats
and in proestrous rats kept under 14L:10D conditions
(LD). Each bar represents the mean determination of
6-8 rats. Vertical lines indicate :1 SEM.

103

ll.
LD

Testosterone (PO/ml serum)

 

TIME

Figure 13. Effect of Constant Light on Plasma Testosterone in
the Morning (AM) and Afternoon (PM).

See Figure 12 for explanation.

104

Not only are minimum levels of plasma estradiol necessary for posi-
tive feedback effects on preovulatory LH release, but dynamic changes
in estradiol may play a significant role as well. Kalra (1975) found
that following the initial "priming" of central sites with low levels of
circulating estradiol during diestrus II, rapid elevations in the ovarian
estrogen secretion between 2300 h of diestrus II and 0300 h of proestrus
facilitated the neural "trigger" of pituitary LH release during the
critical period on proestrus. In addition, Turgeon and Barraclough
(1977) have studied the possible consequences of the abrupt decrease in
estradiol secretion that occurs following the initiation of the gonado-
tropin surge. They found that the pituitary responsiveness to exogenous
LHRH was doubled after decreased circulating estradiol secondary to
ovarian removal. The abrupt decrease in estradiol coupled with the
degree of LHRH presensitization of the pituitary may determine the mag-
nitude of the LH surge. The above types of dynamic changes in estradiol
were not examined in this experiment and thus cannot be ruled out as
possible contributory factors in light induced persistent estrus.

The results of this experiment regarding plasma progesterone levels
also suggest that LL rats may have an insufficient level of progesterone
released as compared to the LD proestrous rat. This is in agreement
with the findings of Takahashi §£_gl, (1977) who also found very low
levels of ovarian vein plasma progesterone but elevated levels of l7-OH—
progesterone in LL rats. These comparative steroid levels (continuous
but low levels of estrogen and low levels of progesterone) are in
accord with the morphological state of the ovary in LL rats since no new

corpora lutea are observed and follicles are at the preovulatory state.

105

Ramaley and Bartosik (1975) found that the rhythmic release of
progesterone and corticosterone which are first seen in immature female
rats at 29 days of age was eliminated by exposing the rats to constant
light from weaning age. Since progesterone levels in their control rats
were greatly reduced by adrenalectomy, they believed the source of the
progesterone rhythm was probably the adrenal.

Nequin and Schwartz (1971), Feder g£_§l, (1971) and Lawton (1972)
suggested that the adrenal secretion of P before the critical period is
involved in facilitating the release of LH on the evening of proestrus.
Progesterone injection into light induced persistent estrous rats re-
initiates cycling (Everett, 1948). It might be implied that if plasma
progesterone levels reached some threshold level in the LL rat, preovu-
latory LH surges should have occurred.

The pattern of changes in plasma testosterone levels in LL rats
appeared to be opposite to that in LB proestrous rats. It might be
implied that plasma testosterone changes in the LL rat are out of
phase with the LD proestrous rat. These are only speculations since
only two time periods were sampled. The role of testosterone in the
normal estrous cycle has not been clearly defined. Mori §£_§l, (1977)
found that injection of antibodies produced against testosterone into
HCG primed rats decreased the number of ova per ovulation to normal.
Androgen has been implicated in the control of follicular maturation
(Louvet 35 gl., 1975). Production of androgen in the follicular fluid
may participate in the regulation of oogenesis in the rat.

Changes in plasma estradiol, progesterone and testosterone secre-

tion seen in LL rats could be a response to constant light or a

106

contributing factor in light induced persistent estrus. Due to the high

degree of integration of the various components of the estrous cycle, it

may be difficult to separate cause from effect unless a thorough time

course of these and other component changes are investigated.

VI. Effect of Constant Light on Hypothalamic Monoamine Metabolism on
Morning and Afternoon Time Periods: Determination of Turnover Index

of Norepinephrine (NE), Dopamine (DA) and Serotonin (5-HT)pin the
AHA and MBH

A. Objective

After exposure to constant illumination (LL), female rats exhi-

 

bit an alteration in gonadotropin release, and enter a persistent
estrous state (Browman, 1937; Hemmingsen.g£_§l,, 1937; Everett, 1942).
The control of gonadotropin release and their corresponding regulation
of ovarian function is in turn influenced by hypothalamic monoamines
(Coppola, 1968; Kamberi ggugl., 1970; Schneider and McCann, 1970).
Kledzik and Meites (1974) have shown that light induced persistent
estrous rats could be induced to resume regular estrous cycles by injec-
tion of L-DOPA and PCA (a drug combination which increases catechol-
amine metabolism and decreases serotonin metabolism). These workers have
hypothesized that constant illumination may induce a persistent estrous
condition by causing a depletion of catecholamine and an excess of sero-
tonin activity.

In order to investigate this hypothesis, norepinephrine (NE),
dopamine (DA) and serotonin (5-HT) turnover index (TI) were measured
in the preoptic-anterior hypothalamic area (AHA) and the medial basal
hypothalamus (MBH) of light induced persistent estrous rats on morning

and afternoon time periods.

107

B. Methods and Materials

 

Mature Sprague-Dawley rats were housed under constant light
conditions for 5-7 weeks. Only those animals exhibiting 10 consecutive
days of vaginal estrus were selected for this experiment. A control
group of rats was housed under 14L:10D lighting conditions. Control
animals used in this study were rats showing a regular 4-day cycle and
selected on the day of proestrus.

Measurement of Hypothalamic Monoamines

 

Catecholamines: Rats were injected intraperitoneally with 200
mg a-methyl-para-tyrosine (aMPT)/kg body weight or its vehicle, 0.85%
NaCl. One hour later, the rats were killed by decapitation, brains
were removed and placed on ice. The medial basal hypothalamus (MBH)
and preoptic-anterior hypothalamic area (AHA) were dissected and frozen
on dry ice. Tissue samples were then homogenized in 20 ul of 0.4 N
perchloric acid (containing 10 mg EGTA/lOO ml).

Dopamine (DA) and norepinephrine (NE) were assayed by the radio-
enzymatic method of Coyle and Henry (1973) as described in the general
Materials and Methods section. The turnover index of catecholamines was
calculated as the percent depletion of NE or DA one hour after a-MPT
injection.

Serotonin: Rats were injected intraperitoneally with 75 mg pargy-
line-HCl/kg body weight or its vehicle, 0.89% NaCl. Thirty minutes
after injection, animals were killed by decapitation, the brains removed,
and the hypothalamus was dissected as above and frozen on dry ice.
Tissue samples were homogenized in 100 pl of 0.1 N HCl (containing 10

mg EGTA/lOO ml). Hypothalamic S-HT was assayed according to the

108
radioenzymatic method of Saavedra 2g 21. (1973). The turnover index of
serotonin was calculated as the percent accumulation of 5-HT following
pargyline injection.

C. Results

Although DA concentrations were significantly greater in the
AHA of LL rats as compared to LD rats (Figure 14) (P<0.05), DA depletion
one hour following o-MPT injection was in most cases not significantly
different between LL and LD rats. The exception to this was found in
the MBH at the AM measurement where DA depletion was higher in LD than
LL rats (57.8:l.9% vs. 46.0:1.9%)(P<0.05). This may suggest that LL
rats have a lower DA metabolism than LD rats in the MBH in the morning.

The AHA norepinephrine concentration was significantly greater in
the AM of LD rats as compared to the PM (Figure 15) (P<0.05). The
opposite trend in AHA NE concentration was seen in LL rats (Figure 15).
Likewise, one hour following a-MPT injection, LD rats showed a greater
AHA NE depletion in the AM than PM (45.9:5.7% vs. 21.3:7.7%) (P<0.05).
In contrast, AHA NE depletion was lower in the AM.than PM (7.5:3.9% vs.
22.2:3.3%) (P<0.05), in LL rats. Furthermore, LD rats showed a greater
NE depletion than LL rats in the AHA (45.9:5.7% vs. 7.5:3.9%) (P<0.05)
in the AM, and in the MBH (55.4:6.5% vs. l6.4:6.0% (P<0.05) at this
time. These results suggest that a decreased NE metabolism exists in
LL rats in the AHA.and MBH during the morning.

The concentration of AHA serotonin was greater in LD than LL rats
in the AM (Figure 17) (P<0.05). MBH serotonin concentration was greater
in LL than LD rats at the PM sampling time (Figure 16) (P<0.05). Thirty
minutes following pargyline injection, LD rats showed a greater AHA

accumulation of 5-HT in the PM than the AM (64.5:9.l% vs. l9.2:3.0%)

Figure 14.

N010

1000 1250

109

DOPAIINI
sellne
L e-mt

a.“ AHA 1750

1200 1500

DION

 

Effect of Constant Light on Dopamine Turnover Index in
Two Areas of the Hypothalamus in the Morning and
Afternoon.

Catecholamine content was measured in the preoptic-
anterior hypothalamic area (AHA) and the medial basal
hypothalamus (MBH) of constant light (LL) rats and pro-
estrous rats kept under 14L:10D (LD) conditions. Morning
(AM) and afternoon (PM) measurements were made 60 minutes
after alpha~methyl paratyrosine (a-MPT) (200 mg/kg) i.p.
or its vehicle 0.89% saline. Turnover index was calcu-
lated as percent depletion of amine after o-MPT. Each
bar represents the mean determination of 6-8 rats.
Vertical lines indicate :1 SEM.

Figure 15.

110

NORIPINIPHIIN!

MBH AHA

2000

NOIO

15m 3

w J

1000

HJ‘M

J
~mu*r

It to ll [0 ll l9 ll LB

Oil”!

3000

2000

1000

Effect of Constant Light on Norepinephrine Turnover
Index in Two Areas of the Hypothalamus in the Morning

and Afternoon.

See Figure 14 for explanation.

Figure 16.

 

lll

SEROTONIN (MBH)

soflne
pctgyline

2000

s g

I.

P
P
U

1
AAA Pan

P
P
0

Effect of Constant Light on Serotonin Turnover Index
in the Medial Basal Hypothalamus in the Morning and
Afternoon.

Serotonin content was measured in the medial basal hypo-
thalamus (MBH) of constant light (LL) rats and proestrous
rats kept under 14L:10D (LD) conditions. Morning (AM)

and Afternoon (PM) measurements were made 30 minutes
following pargyline i.p. (75 mg/kg) or its vehicle 0.89%
saline. Turnover index was calculated as percent accumula-
tion of amine following pargyline. Each bar represents

the mean determination of 6-8 rats. Vertical lines indi-
cate :1 SEM.

112

SERO‘I'ON IN (AHA)

M soline
potgyline

2000

150 '

   
 
   

NO I0

1000

llllllllfll-
II-

P

ID I.
All PM!

P

to

Figure 17. Effect of Constant Light on Serotonin Turnover Index
in the PreOptic-Anterior Hypothalamic Area (AHA) in
the Morning and Afternoon.

See Figure 16 for explanation.

113

(P<0.05). This was also true of PM vs. AM accumulation of 5-HT in the
MBH (94.7:8.0% vs. 57.5:18.4% (P<0.05) of LD rats. No such changes in
5-HT turnover index were seen in PM vs. AM of LL rats. LL rats showed
a greater 5-HT T1 in the AM of the AHA than LD controls (61.8:10.0%
vs. l9.2:3.0%), but a smaller 5-HT TI in the PM of the MBH than LD
controls (54.6:ll.2 vs. 94.7:8.0%). These results suggest an apparent
attenuation of the diurnal 5-HT metabolic rhythm in rats exposed to
constant light.

D. Discussion

 

In general, the present results indicate that a depression in
NE and an increase in S-HT metabolism may occur during the morning in
the AHA of light induced persistent estrous rats. Also a depression in
NE (AM) and 5-HT (PM) metabolism was seen in the MBH together with a
DA (AM) metabolic decrease of questionable significance in this condi-
tion.

Few differences in DA turnover were seen between LL and LD controls
rats in this study. However, DA steady state concentrations were higher
in LL rats than in LD rats. A possible explanation for this may be that
in rats exposed to constant light, the rate limiting enzyme for DA syn-
thesis, tyrosine hydroxylase, may be less sensitive to end produce inhi-
bition, i.e., DA accumulation, together with a decreased release or
decreased reuptake of the amine. In addition, LL may induce a decrease
in the activity of MAO or COMT in this DA system. This selective effect
of constant light on the AHA DA metabolism might also suggest that the
activity of the incerto-hypothalamic DA system in the AHA is affected
by the constant illumination more than the MBH tubero-infundibular DA

system.

114

The LD control rats exhibited a greater morning NE turnover index
than LL rats in either MBH or AHA. Changes in hypothalamic concentra-
tion and metabolism of NE have been reported to occur during stages of
stimulatory steroid feedback. On proestrus of the estrous cycle, an?
terior hypothalamic (Stefano and Donnoso, 1967; Coppala, 1969) and median
eminence (Selmanoff g£_§l,, 1976) NE concentration and whol brain NE
turnover increase (Zacharek and wurtman, 1973). During the first
estrous cycle, hypothalamic NE turnover increases during early proestrous
and decreases in late proestrous and estrus (Advis ggflgl., 1978). The
data of this experiment on AM vs. PM NE turnover index in LD rats is in
agreement with the above reports. Most studies indicate that NE has a
stimulatory effect on LH secretion (Sawyer, 1975; Krieg and Sawyer, 1976;
Kalra g£_§l,, 1972). The absence of this stimulatory hypothalamic input
in the constant light exposed animal may contribute to the persistent
estrous condition in which the stimulation for ovulation, i.e., LH
release, is also missing.

Comparing the AHA diurnal rhythmi of NE turnover index in the LL
rats with the LD controls, it appears that constant light results in a
phase shift of the NE metabolic rhythm during the day (Figure 15).
Since measurements of NE TI were made at only two time periods, this
hypothesis needs to be tested further at additional time periods. Per-
haps the light-dark cycle synchronizes central neuronal activity rhythms
which could involve NE turnover. Induction of an IR surge during the
estrous cycle may depend on the synchronous activity of multiple monoami-
nergic neurons. The absence of the external signal entraining these

rhythms may result in failure of a preovulatory LH surge to occur.

115

The diurnal rhythm in serotonin turnover index (low in the AM and
high in the PM) seen in both areas of the brain in LD rats was not
apparent in the LL rats in this study. These data are in agreement
with the observations of Meyer and Quay (1976a) who reported that a
diurnal variation in 5-HT uptake occurs in the suprachiasmatic nuclear
region (SNR) of male and female rats $2;21££29 with a peak during the
late light to early dark period. They suggested that this increase in
SNR uptake of 5-HT may serve to limit free 5—HT and its inhibitory
effects on the gonadotropin release hormone system. In addition, Meyer
and Quay (1976b) have found that rats exposed to continuous light show
no significant difference in serotonin uptake by the SNR at 1000 or
2200 h. In the present study, the AHA S-HT turnover index was greater
in the LL than LD rat in the AM. In contrast to the LD rat, the LL rat
showed no change in AM vs. PM serotonin turnover index. This elevation
in morning serotonin TI and/or arrhythmic diurnal variation in the LL
rat may have an inhibitory influence on LH release.

Kueng SE 31. (1976) examined S-HT concentrations in 18 brain
regions during the estrous cycle of the rat. They found that in the
lateral preoptic area, the 5-HT concentration was elevated in the
morning of proestrus and then decreased in proestrous afternoon, estrus
and metestrus. The LD rat serotonin concentrations in the AHA showed the
same pattern of changes during the day in these studies. Kueng 22 El.
(1976) proposed that there may be increased release of 5-HT in the
afternoon of proestrus. In support of their hypothesis, these studies
also suggest an increased 5-HT turnover in proestrous afternoon, but a

constant high level of 5-HT turnover during the day in LL rats.

116

In constrast to the AHA, LL rat 5-HT turnover index was lower than
that of LD rats in the MBH. These differences in S—HT metabolism in
two areas of the brain in the same type of animal appear very unusual
but are not necessarily mutually exclusive. Just as different areas of
the brain may have opposite effects on a physiological function, the
same neurotransmitters in those areas could have opposite effects as
well. Perhaps the AHA 5-HT center is inhibitory to LH release during
most of the estrous cycle while the MBH 5-HT center is stimulatory.
Alternatively, MBH 5-HT metabolism may not play any significant role
in control of the preovulatory LH surge.

In any brain region, the metabolism of a neurotransmitter is in-
fluenced by the activity and metabolism of other neurotransmitters,
and by the activity of free amino acids which may act both as putative
neurotransmitters and as precursors for monoamine transmitters. It
is therefore difficult to ascribe abnormalities in brain function to
alterations in a single specific neurotransmitter substances inasmuch
as overall brain activity ultimately depends on the sum of changes in
all neurotransmitters.

It has been postulated that the hypothalamus exerts a dual control
over ovulation--its inhibitory influence being transmitted via a 5-HT
pathway and a stimulatory one through a catecholaminergic (CA) path—
way. It is believed that the balance between these contrasting in-
fluences determines the occurrence of ovulation--a critical balance
in favor of the CA pathway promoting ovulation, and the opposite

inhibiting it (Labhsetwar and Zolovick, 1973).

117

There appear to be similarities in neuroendocrine changes occurring
in the reproductive state of constant estrus as induced either by old
age or constant light in the rat. In general, the results of these
studies suggest that a depression in AHA NE metabolism and MBH 5-HT
and NE metabolism but an increase in AHA 5-HT metabolism (AM) may occur
in light induced persistent estrus. Huang (1978) showed that old (20
month) female rats in constant estrus show a lower turnover index of
NE in the AHA than younger regular cycling animals on proestrus or
estrus. Previously it has been observed that chronic stimulation of
catecholamine or depression of serotonin metabolism in old female rats
in constant estrus results in improved reproductive function (Clemens
g£_§i,, 1969; Quadri g£_§1,, 1973). Finally, Everett (1970) has shown
that when old constant estrous rats were placed in an environment with
less than 10 hours of light, regular cycling resumed. Although it
would be tempting to speculate that the light induced persistent estrous
condition may be a useful model in which to study the effects of aging
on neuroendocrine control of reproduction, further work is necessary

to evaluate the possible relationship between these two phenomena.

DISCUSSION

This report explains some of the changes which occur at various
levels of the hypothalamo-hypophysial-gonadal axis in the light
induced persistent estrus rat. A common neuroendocrine change seen in
various states of noncyclicity (such as light induced persistent estrus)
is the absence of a preovulatory LH surge. Therefore, attention was
focused on those processes directly or indirectly involved in initiating
and maintaining this surge and considers possible mechanisms for the
effect of constant light on reproductive function in the rat.

Based on experiments where the daily administration of a barbitu-
rate delayed ovulation by 24 h intervals, Everett and Sawyer (195) con-
cluded that preovulatory secretion of LH in the rat displayed in a
definite 24 h periodicity. Since the time of the preovulatory surge
of LH in plasma was advanced or delayed in response to corresponding
shifts of the photoperiod (Everett, 1970; Blake, 1975), it became obvious
that the 24 h periodicity was synchronized to the light-dark schedule.
The fact that cyclic ovulation occurred for long periods in rats kept in
continuous darkness suggests that daily light-dark cues are not essen-
tial for maintaining the cyclic mechanism of the LH surge and suggests
the synchronizer may be endogenous. In the absence of a light-dark
cycle, changes in temperature or humidity (Wurtman, 1967) may furnish
sufficient stimulus for establishing the phase of the estrous cycle.

The possibility of a spectrum or hierarchy of environmental cues

118

119
complicates claims of inherent rhythmicity of reproductive functions.
McCormack and Sridaran (1978) found that after exposing rats which had
been allowed to "free run" in an environment of very dim constant light,
to a light:dark regimen, the rats resynchronized to the new lighting
schedule. This may suggest that the lightzdark regimen may "cue" some
endogenous synchronizer for LH release or that it is a stronger synchro-
nizer than some other environmental cue.

Whereas rhythmic changes in environmental lighting exert a synchro—
nizing effect on estrous cycles, conditions of continuous light or dark
may result in aberrant vaginal cyclicity. Rats under constant illumina-
tion, with possible strain differences in sensitivity, commonly show
persistent vaginal cornification (Hoffmann, 1973). These distrubances
in the estrous cycle resulting from constant illumination or dark
suggest that light is more intimately involved in mechanisms controlling
gonadotropin secretion than is generally implied in its accepted role as
synchronizer or monitor of reproductive functions in mammals (Critchlow,
1963).

Among the effects of constant light on reproduction is the eleva—
tion in serum FSH and Prl levels while serum LH levels are decreased or
unchanged. In accordance with previous studies (Kledzik and Meites,
1974) no preovulatory LH surge was seen in LL rats in Experiment 1.
Morning serum FSH levels were not elevated in LL rats but rose in the
afternoon, just as in LD proestrous rats. This differential effect of
constant light on gonadotropin release is interesting in that it
supports the theory for separate hypothalamic releasing hormones for

LH and FSH. It also suggests that constant light may be selectively

120
inhibiting neuronal areas which are more closely involved with LH
release.

Administration of testosterone proprionate (TP) to 5-day old female
rats produces a syndrome characterized by anovulatory ovaries and
vaginal cornification (Barraclough, 1961). Barraclough (1966) also has
demonstrated that neonatal TP treatment deleteriously affects neural
mechanisms responsible for the cyclic discharge of LH. These effects
are similar to those found in constant light induced persistent estrus.
Recent studies by Chappel and Barraclough (1976) have shown that electro-
mechanical stimulation of the medial preoptic area (MPOA) induces the
release of both LH and FSH whereas a selective release of pituitary FSH
can be elicited by dorsal anterior hypothalamic area (DAHA) stimulation.
Chappell and Barraclough (1976b) have also shown that neonatal steroid
exposure does not affect those DAHA components involved in FSH release
but significantly less LH was released after MPOA stimulation in
these animals. Perhaps constant light is selectively affecting these
same hypothalamic areas in a similar pattern.

The present studies have ruled out the possibility of a decreased
pituitary sensitivity to GnRH as a probable cause for absence of a
preovulatory LH surge in the LL rat. On the contrary, the LL rat pi-
tuitary was found to have comparable sensitivity to GnRH as the LD pro-
estrous rat pituitary as reported by Fink (1975). This increased pi-
tuitary sensitivity would in turn explain the greater effectiveness
with which ovulation occurs in LL rats in response to mating (Smith and
Davidson, 1974). This ability to ovulate in response to mating, to a
brief period of darkness (Takahashi g£_§l,, 1977) or even to stress

(Smith and Davidson, 1974) also suggests that LHRH is still synthesized

121
in the hypothalamus of LL rats and furthermore can be released to
affect pituitary LH release under these conditions. In an LL rat not
exposed to these stimuli, however, spontaneous release of LHRH into the
pituitary portal system would not be expected to occur. The results
of hypothalamic LHRH content measurements in the LL rat (Experimental
III) suggest that this may indeed by the case.

One of the factors involved in the physiolOgical regulation of
spontaneous and cyclic LHRH release is the sensitivity of the hypothala-
mus to positive and negative feedback by estrogen (E) and progesterone
(P). Hoffmann (1973) has postulated that in light induced persistent
estrus, just as in persistent estrus due to old age, the hypothalamic
sensitivity to estrogen positive feedback may be decreased. Huang 22
‘31. (1978) have shown that a decreased positive feedback sensitivity to
estrogen occurs in old (24 month) constant estrous rats. Similarly,
neonatal androgen induced persistent estrous rats fail to demonstrate
positive feedback release of LH in response to estrogen priming (Mennin
and Gorski, 1974). The same effect was seen in light induced persistent
estrous rats in Experimental section IV in this thesis. Furthermore,
in all three of the above types of persistent estrous states, estrogen
priming followed by progesterone administration was found to be effec-
tive in inducing a positive feedback release of LH. It would appear
then that progesterone was able to overcome whatever changes occurred
in hypothalamic positive feedback sensitivity to estrogen in these
persistent estrous states. Presumably then, if progesterone were present
in sufficient levels at the proper time, spontaneous LH release should
occur in these animals. Exogenous progesterone administration at

appropriate times during the 4 or 5 day estrous cycle will advance

122
ovulation 24 hours in intact rats (Everett, 1948; Brown-Grant, 1969).
Progesterone synergizes with subthreshold dosages of ethanol estradiol
to induce an ovulatory LH surge in castrate female rats (Swerdloff £1_
21,, 1972).

Ovarian progesterone secretion does not increase prior to the pro-
estrous release of gonadotropin (Barraclough g£.§1,, 1971; Piacsek g;
31,, 1971). Consequently, if this steroid synergizes with estrogen to
facilitate this event, it must originate from some extra-ovarian source.
Feder g£_§1, (1968) have demonstrated that the rat adrenal glands secrete
substantial amounts of progesterone. Furthermore, in a recent investiga-
tion Mann and Barraclough (1973) have shown that a diurnal rhythm exists
in adrenal progesterone secretion during the rat estrous cycle; being
high during the early morning hours (0100-0500 hr) and then falling to
a nadir between 1000 and 1400 hr. A possible role of adrenal proges-
terone in the regulation of LH secretion on proestrus in the rat has been
proposed by several investigators (Nequin and Schwartz, 1971; Feder'gg
31,, 1971; Lawton, 1972). Mann and Barraclough (1976) have reported
that progesterone was necessary in order to stimulate an LH rise in EB
primed ovariectomized-adrenalectomized rats. Moreover, these investi-
gators have prOposed that adrenal progesterone secretion is responsible
for synchronizing the timing of the LH surge and thus the critical
period. Since adrenal progesterone secretion is controlled by ACTH
(Resko, 1968) and release of this trophic hormone is synchronized by
the light-dark cycle, then perhaps the timing of the critical period
for LH release actually is indirectly regulated via circadian adrenal

progesterone rhythms.

123

Persistent estrus induced by old age or constant light is accom-
panied by an apparent cessation in the rhythm in steroid secretion.
Huang g1 El: (1978) observed low and arrhythmic secretion of estradiol
and progesterone secretion in old constant estrous rats as compared to
young regular cycling rats. Takahashi g£_§1, (1977) reported that con-
stant light induced persistent estrous rats had plasma estradiol levels
which were intermediate between proestrus and diestrus levels but also
very low levels of progesterone. Constant light has been shown to
abolish the rhythm of corticosterone secretion, which is presumably
a response to a change in ACTH release in this condition (Critchlow,
1963; Takahashi g£_§1,, 1977). In accordance with the results of Taka-
hashi g£_§1,, 1977), intermediate estradiol levels and low levels of
progesterone were seen in light induced persistent estrous rats in
Experimental section V of this thesis. Since both adrenal cortico—
sterone and progesterone release respond to ACTH secretion, which is in
turn synchronized to the light-dark cycle, constant light might be
expected to result in a disturbance in the progesterone rhythm. The
low levels of progesterone seen in light induced persistent estrous
rats in Experimental section IV may be one of the major contributing
factors inducing the persistent estrous condition. Progesterone injec-
tion has been reported to induce cycling in LL rats (Everett, 1948)
and old constant estrous rats (Huang g£_§1,, 1976). Perhaps under con-
ditions such as constant light, hypothalamic sensitivity to positive
feedback by estrogen is decreased and therefore the facilitatory
support of progesterone becomes necessary although absent in this

condition.

124

The sensitivity of the hypothalamus to positive and negative feed-
back by steroids may be interpreted mechanistically as the effective-
ness with which steroids affect hypothalamic monoaminergic neuronal
activity. The activity of these neurons directly or indirectly affects
GnRH neurosecretory neurons and their corresponding release of gonado-
tropins. Kalra 35‘51. (1971) have shown that the stimulatory effects
of progesterone on LH and FSH release involve noradrenergic neurons.

In Experimental section VI, it was seen that light induced persistent
estrous rats showed a decreased morning NE turnover index in the anterior
hypothalamic area and medial basal hypothalamus as compared to LD pro-
estrous controls. Also, morning 5—HT turnover index was higher in

the AHA of LL rats along with an apparent loss of daily 5-HT metabolic
rhythm. These results support the hypothesis of Kledzik and Meites
(1974) that LL may result in decreased catecholamine and increased sero-
tonin metabolism. They were able to reinitiate cycling in LL rats by
administration of catecholamine precursor and 5-HT inhibitor drugs.

Not unlike light induced PE, Huang (1978) demonstrated that old constant
estrous animals showed a decreased hypothalamic NE turnover index as
compared to young proestrous or estrous rats.

NE has been reported to stimulate release of gonadotropins (Kalra
and McCann, 1973b) and 5-HT has been reported to be inhibitory (Kamberi
.ggug1., l970b) or stimulatory to LH release (Takahashi 91 31., 1973).
Therefore an alteration in the function of these hypothalamic neuronal
systems in response to constant light exposure, as seen in Experimental
section VI, could result in the blocking of the preovulatory LH surge.
It is possible that constant light could directly affect the function

of these neuronal systems and thus affect gonadotropin release.

125
Alternatively, it is also possible that constant light could alter ACTH
release rhythm and its corresponding influence on adrenal progesterone
rhythm which in turn could alter activity of hypothalamic neuronal
systems to block preovulatory LH release. Regardless of which of these
routes constant light takes to affect gonadotropin release, its initial
effect on reproduction appears to be at the level of the brain.

Reviewing the data presented in this thesis, no clear hypothesis
regarding a cause and effect relationship between constant light and
its effect on reproductive cyclicity can be made. Although some
mechanisms for this effect have been ruled out in these experiments,
only correlations between hypothalamic and hormonal changes in light
induced persistent estrous can be made at this time. Due to the complex
integration of components involved in maintaining this "cycle", it may
be difficult to distinguish cause from effect after persistent estrus
is established. Nevertheless, these experiments have shed some light
on which pieces of this puzzle are of importance and will hopefully
stimulate further inquiry into the etiology of light induced persistent
estrus.

Some effects of light and photOperiod appear to have a direct
influence on human reproductive cycles (Dewan, 1967) and on domestic
animals important as a human food source (Peters ggflg1., 1978). Investi-
gation of the full potential of light in these circumstances could be
very beneficial. Since it would be important to know about the mecha-
nisms by which light may induce these responses, a study of the mecha-
nisms in light induced persistent estrus may offer some clues about

the mechanism involved in the above light effects as well.

APPENDICES

APPENDIX A

Coyle and Henry Catecholamine Assay Procedure

Homogenize pieces of brain tissue in desired volume of 0.4 N per-
chloric acid (plus 10 mg% EDTA) using matched glass microhomogeni-
zers (Micrometric Instruments, Cleveland, Ohio).

Transfer homogenate to microcentrifuge tubes (Kew Scientific, Inc.,
Columbus, Ohio) and centrifuge for 45 sec in a microcentrifuge
(Coleman Instruments, Oak Brook, Illinois).

Transfer 10 u of supernatant (or of working NE and DA standard
solution) to glass culture tubes and add 25 ul of the following
mixture:

 

Reagent Proportion
20 mM EGTA-Na salt (0.760 gm/lOO m1 1
H 0 and pH to 7.2)

2

Pargyline Solution (to 4 mg pargyline add 25 ul
B-mercaptoethanol and 225 pl H20) 1

l M Tris base (with 3 mM MgCl ) (to 6.05 gm

Tris add 50 ml H20 plus 30.5 %g MgClz) 6.5
S-adenosyl-l-methionine(Methyl-3H) [11.6 Ci/m

mole in Sulfuric acid:ethanol solution (90:10,

v:v), pH 1-3] _ 3'0
Catecholamine-o-methyl transferase (COMT, par-

tially purified by the method of Nikodijevic

21-31,, 1970) 2.5
1 mM sodium phosphate buffer 2.5

Incubate for 40 min at 37°C.

Add 30 ul of mixture of 5 volumes 0.45 M borate buffer (pH 10.0)
and 1.0 volumes of carrier methoxyamine mix prepared as follows:

add 5.0 m1 H20 to the following salts:

50 mg 3~methoxytyramine, 50 mg DL—metanephrine, 50 mg
DL-normetanephrine and 5 mg Na-bisulfite

126

10.

ll.

12.

13.

14.

127

Add 500 pl to toluene:isoamyl alcohol solution (3:2, v:v), vortex
for 30 sec and centrifuge for 5 min at 3,000 RPM (RC2-B, Sorvall,
Dupont Instruments, Newtown, Connecticut).

Transfer 400 pl of organic phase to conical centrifuge tubes con—
taining 400 pl borate buffer (pH 10.0), vortex for 30 sec and
centrifuge at 5/7 speed in IEC clinical centrifuge (International
Equipment Co., Needham Hts., Mass.).

Transfer 300 pl of organic phase to conical centrifuge tubes con-
taining 500 pl of 0.1 N HCl, vortex for 30 sec and centrifuge as
in step 7.

Aspirate organic phase.

To remaining aqueous phase add 7 m1 toluene:isoamyl alcohol (3:2,
v:v), vortex, centrifuge as in step & and discard organic phase.

To remaining aqueous phase neutralize with 500 pl of 0.5 M sodium
phosphate buffer (pH 7.5), add 50 ul of 3% sodium metaperiodate
wait 2 min and add 50 pl of 10% glycerol.

Add 10 m1 toluene, vortex for 30 sec, centrifuge as in step 7.

Transfer 9 ml of organic phase to conical centrifuge tubes con-
taining 1 m1 of l N NaOH for final extraction of NE metabolites.
Vortex for 30 sec, centrifuge and discard organic phase. Add
100 pl glacial acetic acid, 10 ml Scintiverse (Fisher Scientific,
Livonia, Michigan) and transfer to 20 ml glass scintillation
vials for counting.

From the remaining aqueous phase of step 12, the residue toluene
is aspirated, 500 p1 of l M borate buffer is added and tubes are
vortexed. Add 8 ml of toluene:isoamyl alcohol (3:2, v:v), vortex
and centrifuge. 0.6 ml of the organic phase is added to 10 ml of
Scintiverse in 20 ml glass scintillation vials and counted for
dopamine.

APPENDIX B

Ben—Jonathan and Porter Catecholamine Assay Procedures

 

Same as step 1 in Appendix A.
Same as step 2 in Appendix A.
Transfer 10 pl of supernatant (or of working NE and DA standard

solutions) to conical centrifuge tubes and add 25 pl of the
following mixture:

 

Reagent Pppportion
20 mM EGTA-Na salt (0.760 gm/lOO m1 H20 and
pH to 7.2) l

Pargyline solution (to 4 mg pargyline add 25 pl
B-mercaptoethanol and 225 p1 H20) 1

l M Tris base (with 3 mM MgC12) (to 6.05 gm
Tris add 50 m1 H20 plus 30.5 mg MgClz) 6.6

S-adenosyl methionine (Methyl-3H) (11.6 Ci/m
mole diluted 1:3.5 with H20) 3.0

Catecholamine-o-methyl transferase (COMT;
partially purified by the method of Nikodijevic
g£_a1,, 1970) 5.1

Incubate for 60 min at 37°C.

Add 30 pl of 0.45 M borate buffer (pH 10.0) and 5 pl of carrier
methoxyamine mix (50 mg 3-methoxytyramine, 50 mg DL-metanephrine
and 50 mg DL-normetanephrine; 10 mg of each amine/ml of 0.1 N
HCl). Add 500 p1 of toluene:isoamyl alcohol (3:2, v:v), vortex
for 30 sec, and centrifuge at 5/7 speed on IEC clinical centri-
fuge (International Equipment Co., Needham Hts., Mass.).

Transfer 400 p1 of organic phase to conical centrifuge tubes

containing 40 pl of 0.1 N HCl. Vortex for 30 sec and centrifuge
as in step 5. Carefully aspirate organic phase.

128

10.

129

Apply 25 pl of acid phase to LQ-6O silica gel plates previously
spotted with 5 pl of carrier methoxyamine mix. Allow plates to
dry.

Place plates in thin-layer chromatography tanks containing chloro-
form, ethanol and methylamine (40:18:5 by volume). Allow plates
to run 1-1/2 to 2 h and remove from tank to allow for drying.

Visualize and outline spots under ultraviolet light.
Scrape plates and place scrapings into scintillation vials con-

taining 1.0 ml of ethylacetate, acetic acid and H20 (3:3:1 by
volume) and shake for 30 min. Add 10 ml of Scintiverse and count.

APPENDIX C

Serotonin Microassay

 

Assay:
l. Homogenize tissue in 100 pl 0.1 N HCl. (with 10 mg EGTA/lOO m1)
2. Centrifuge 6,000 RPM (RC2-B) 5 min.
3. Add 10 pl supernate to 5 ml disposable culture tube containing

10 pl P04 buffer - NaOH.

4. Add 20 pl of a mixture of equal parts of Acetyl CoA, 1 mg/ml (PO4

buffer) + NAT.

Incubate 37°C/30 min.

. Add 25 pl of a mixture containing 14.5 parts PO

HIOMT, 0.5 parts SAM-H3.

7. Incubate 37°C/10 min.

8. Stop reaction: note - stop ASAP. Place on ice and add 100 pl
Borate Melatonin (5 mg/ml).

9. Extract in 2 ml toluene.

10. Vortex 30 sec.

11. Spin at 6,000 RPM/5 min (RCZ-B).

12. Aspirate 1.5 ml supernate (organic phase) and transfer to a scin-
tillation vial containing 2 ml fresh toluene.

13. Dry overnight at 80°C (hot plate - time - 25%) in hood (in pan).

14. Add 1 ml EtOH + 10 ml scintiverse.

15. Count.

O‘U‘

4 buffer, 10 parts

Std.:

 

Serotonin 10 pg/ml 0.1N HCl (free base). Weigh out 21.48 pg serotonin
creatinine sulphate/m1 0.1N HCl.

Working Soln. Take 5 pl stock serotonin:
5 pl -qs + 1 ml (0.5 ng/lO pl)
10 pl -qs +'1 ml (1.0 ng/lO pl)
20 pl -qs + 1 ml (2.0 ng/lO pl)

 

Reagents:

PO, buffer 0.2M pH 7.9 Titrate dibasic (2.84 g/100 ml) with Monobasic
I (2.76 g/100 ml) such that pH = 7.9

 

Borate Melatonin ‘Mix 9 parts Borate 0.5M pH 10 (6.2 g/200 ml H20
brought to pH 10 with 5N NaOH pellets) with 1 part Melatonin 5 mg/ml
(50 mg Melatonin/2.5 ml EtOH....bring up to 10 ml with H20).

 

130

131

0.1N HCl (containing 10 mg EDTA/lOO ml).

PO, buffer - NaOH (22.5 ml PO buffer + 2.5 ml 1N NaOH).
—1-9

4
Scintiverse (Fisher).

APPENDIX D

Steroid Radioimmunoassaprrotocol

Extraction of Serum.

lO.

Pipette desired amount of serum into 18x150 mm test tubes.
Add 1 ml ether/0.1 ml serum to serum tubes.
Vortex (30 sec).

Place tubes in dry ice-ethanol bath until serum fraction is
frozen.

Decent ether fraction (which now contains the extracted
steroids) into 16x125 mm test tubes.

Dry ether fraction with air.
Wash tube walls with ether (about 1 ml) and dry with air.
Add 1 ml PBS-gel to tubes when completely dry.

Vortex tubes for 30 seconds and allow to stand at room tempera-
ture for 30 min.

From the 1 m1 PBS-Gel, pipette desired dilution into assay
PBS-Gel so that the total volume is .5 ml.

Recovery, Tracer and Solvent Blank Tubes.

1.

2.

Tracer tubes: add 100 pl labeled steroid to scintillation
vials, then add 100 pl PBS-Gel and 5'ml counting fluid.

Recovery tubes: add 100 p1 labeled steroid to serum samples
not to be assayed and extract as above (steps 1-9, use 200
pl).

Solvent blank: extract distilled water (1 ml) as in steps 1-9.

132

133

Standard (Estradiol, Progesterone and Testosterone).

l.

2.

Standards are made in 100% EtOH (5 pg/pl).

Standard curve: 2.5, 5, 10, 20, 40, 60, 80, 100, 200, 400,
800 pg.

Dry standard in air.

Add 0.5 m1 PBS-Gel to all standard tubes. Add 0.6 m1 PBS-Gel
to total count and non-specific binding tubes.

Hormone Assay.

1.

Add 100 pl antibody to assay tubes and standard, except TC and
NSB tubes.

Vortex and incubate at room temperature for 30 minutes.

Add 100 pl of labeled steroid to all tubes.

Incubate at 4°C overnight.

Add 200 pl charcoal dextran on ice (vortex).

Incubate 10 minutes on ice.

Centrifuge at 1000 g in 4°C.

Take 0.5 ml supernatant and add to 3 ml scintillation fluid.

Count.

Name:

Date of Birth:

 

Place of Birth:

 

Sex:

Marital Status:

 

Present Address:

 

Education:

Positions Held:

 

Research Interests:

 

CURRICULUM VITAE

 

Robert John Miodusczewski
November 30, 1947

Toledo, Ohio

Male

Single

Department of Physiology, Michigan State
University, East Lansing, MI 48824

University of Toledo, 1969, 8.8., Biology

University of Toledo, 1971, M.S., Biology
(C.J.V. Smith)

Michigan State University, 1978, Ph.D.,
Physiology (J. Meites)

1) Graduate Research Assistant, Physiology
Department, Michigan State University, 1975
to present.

2) Graduate Teaching Assistant, Physiology
Department, Michigan State University, 1974
to 1975.

3) Research Technician (Sgt., United States
Army), Biology Department, Division of
Medicinal Chemistry, also Neuroendocrinology
Department, Division of Neuropsychiatry,
Walter Reed Army Institute of Research,
Washington, D.C., 1972 to 1974 (Honorable
Discharge).

4) Research Assistant, Biochemistry Department,
Medical College of Ohio at Toledo, June 1971
to October 1971.

5) Graduate Teaching Assistant, Biology Depart-
ment, University of Toledo, September 1969
to June 1971.

Mechanisms of hypothalamic and extrahypothalamic
control of anterior pituitary hormone secretion,
particularly the role of biogenic amines in re-
sponse to various environmental stimuli, reproduc-
tive states, and the aging process.

134

135

 

Research Techniques: Radioimmunoassay; LH, FSH, Prl, TSH, CH, LHRH
estradiol, progesterone and
testosterone.

Radioenzymatic assay; NE, DA, 5-HT.

Stereotaxic and general endocrine surgical pro-
dures.

Column and thin layer chromatography.

Publications:

 

Mioduszewski, R., L. Grandison and J. Meites. Stimulation of prolactin
release in rats by GABA. Proc. Soc. Exp. Biol. Med. 151: 44-46,
1976.

 

Mioduszewski, R., H.T. Chen, C.A. Hodson, R.W. Steger and J. Meites.
Decreased hypothalamic biogenic amine turnover index by constant
illumination. Fed. Proc. (Abstract presented at the 62nd Annual
FASEB Meeting, Atlantic City, N.J., L978.

 

Simpkins, J.W., J.F. Bruni, R.J. Mioduszewski and J. Meites. Serum and
pituitary TSH and response to TRH in developing male and female
rats. Endocrinology 28; 1365-1369, 1976.

 

Hall, T.R., R. Mioduszewski, C.A. Campbell, L. Grandison and J. Meites.
Effects of Gamma-aminobutyric acid on prolactin release from rat
pituitaries incubated 12 vitro. IRCS J. Med. Sci. 5; 112, 1977.

 

Lu, K.H., H.H. Huang, H.T. Chen, M. Kurcz, R. Mioduszewski and J. Meites.
Positive feedback of estrogen and progesterone on LH release in
old and young rats. Proc. Soc. Exp. Biol. Med. 154: 82-85, 1977.

 

Bruni, J.F., R,J. Mioduszewski, L.J. Grandison, J.W. Simpkins and J.
Meites. Effects of cholinergic and GABA-ergic drugs on serum
growth hormone (GH) in male rats. Fed. Proc. 36: 323, 1977.

Campbell, C.A., C.A. Hodson, R. Mioduszewski and J. Meites. Increased
estrogen suppression of LH in starved, ovariectomized rats.
Endocrine Society Proceedings, 1977 (Abstract).

Hodson, C.A., R. Mioduszewski and J. Meites. Effects of adrenergic
agonists and antagonists on growth of DMBA-induced summary tumors
and serum prolactin levels. ASCO Abstracts 18: 636, 1977 (Abstracts).

Bruni, J.F., J.W. Simpkins, R.J. Mioduszewski and J. Meites. 1p_vivo and
13 vitro stimulation of LH release by thyroid hormones. (Submitted
to Life Sciences).

 

Hodson, C.A., R. Mioduszewski, and J. Meites. Effects of catecholaminer-
gic and anti-catecholaminergic drugs on growth of carcinogen-induced
mammary tumors in rats. (Submitted to Proc. Soc. Exp. Biol. Med.)

136

Mioduszewski, R., H.T. Chen, C.A. Hodson, R.W. Steger and J. Meites.
Changes in hypothalamic biogenic amine turnover index in light
induced persistent estrus. (In preparation).

Campbell, C.A., C.A. Hodson, R. Mioduszewski and J. Meites. Decreased
threshold for negative feedback action of estradiol on LH secretion
during starvation. (In preparation).

Campbell, C.A., R. Mioduszewski, C.A. Hodson and J. Meites. Normal LH
surge induction by progesterone in starved ovariectomized rats.
(In preparation).

Mioduszewski, R. Hypothalamic control of carbohydrate metabolism in the
diabetic rat: blood glucose and plasma growth hormone levels
following bilateral electrolytic lesions of the ventromedial hypo-
thalamus. Masters Thesis, University of Toledo, 1971.

Mioduszewski, R. The effect of constant illumination of the hypothalamo-
hypophysial—gonadal axis of the female rat. Ph.D. Dissertation,
Michigan State University, 1978.

BIBLIOGRAPHY

BIBLIOGRAPHY

Adams, J.R., P.M. Daniels and M.M.L. Prichard: Distribution of hypo-
physial portal blood in the anterior lobe of the pituitary gland.
Endocrinology 75:120, 1964.

 

Adler, N.T. and D. Bell: Constant estrus in rats: Vaginal reflexive
and behavioral changes. Physiol. Behav. 4:151-153, 1969.

 

Advis, J.F., J.W. Simpkins, C.H. Chen and J. Meites: Relation of
biogenic amines to onset of puberty in the female rat. Endocri-
nology 103:11—16, 1978.

Aghajanian, G.K., and I.M. Asher: Histochemical fluorescence of raphé
neurons: Selective enhancement by tryptOphan. Science 172:1159-
1161, 1971.

Aghajanian, G.K. and F.E. Bloom: Electron microscopic localization of
tritiated NE in rat brain: Effect of drugs. 1, Pharmacol. Exp.
Ther. 156:407-416, 1967.

 

Aiyer, M.S., G. Fink and F. Greig: Sensitivity of the anterior pituitary
gland to lueinizing hormone releasing factor (LRF) during pro-
oestrus in the rat. g, Physiol. 231:32P-34P.

Allardyce, J., J. Aldous, N. Cooper, J. Pratt and E. Sutherland: Effects
of visible radiation upon albino rats. Am, 1, Physiol. 137:761-768,
1942.

Alleva, J.J., J.G. Overpeck and E.J. Umberger: Effect of tranglycypro-
mine and iproniazid on brain amine levels and ovulation in the
golder hamster. Life Sci. 5:1557-1561, 1966.

Alleva, J.J., M.V. Waleski and F.R. Alleva: The zeitgeber for ovulation
in rats: Non-participation of the pineal gland. Life Sci. 9:241-
246, 1970.

Alpert, L.C., J.R. Brower, Y.C. Patel and S. Reichlin: Somatostatinergic
neurons in the anterior hypothalamus: Immunochemical localization.
Endocrinology 98:255-258, 1976.

 

Amoroso, E.C. and F.H.A. Marshall: External factors in sexual periodi-
city. In: Marshall's Physiolggy pf Reproduction (3rd ed.), edited
by A.S. Parkes. London: Longmans, Vol. I, part II, p. 707-831,
1960.

 

137

138

Andén, N.E., A. Carlsson, A. Dahlstrdm, K. Fuxe, N.A. Hillarp and K.
Larson: Mapping out of catecholamine and 5-HT neurons innervating
the telencephalon and diencephalon. Life Sci. 4:1275-1279, 1965.

Andén, N.E., A. Dahlstr6m, K. Fuxe, K. Larson, L. Olson and U. Unger-
stedt: Ascending monoamine neurons to the telencephalon and dien-
cephalon. Acta Physiol. Scand. 67:313-326, 1966.

 

Anderson, E. and W. Haymaker: Breakthroughs in hypothalamic and pitui-
tary research. In: Progress 1n_Brain Research, edited by D.F.
Swaab and J.F. Schade, Vol. 41, p. l, Elsevier, New York, 1974.

 

D'Angelow, S.A. and A.S. Kravatz: Gonadotrophic hormone function in
persistent estrous rats with hypothalamic lesions. Proc. Soc. Exp.
Biol. 104:130-133, 1960.

 

Aprison, M.H. and J.N. Hingtgen: Serotonin and behavior: A brief
summary. Fed. Proc. 31:121-129, 1972.

Arbuthnott, G.W., T.J. Crow, K. Fuxe, L. Olson and U. Ungerstedt: Deple-
tion of catecholamines 1n_vivo induced by electrical stimulation of
central monoamine pathways. Brain Res. 24:471—483, 1970.

Arimura, A. and A.V. Schally: Methods for estimating the biologic acti-
vity of the luteinizing hormone and follicle stimulating hormone-
releasing hormone. Methods Enzymol. 37, pt 13:233-238. 1975.

 

Artunes-Rodrigues, J. and S.M. McCann: The effect of suprachiasmatic
lesions on the regulation of LH secretion in the female rat.
Endocrinolpgy 81:666-670, 1967.

 

Aschner, B.: Uber die Funktion der Hypophyse. Arch. Ges. Physiol.
146:1-146, 1912.

 

Asdell, S.A.: Patterns 21_Mammalian Reproduction. Ithaca, N.Y.:
Cornell University Press, 1964.

 

Austin, C.R. and A.W.H. Braden: Time relations and their significance
in the ovulation and penetration of eggs in rats and rabbits.
Australian 1, Biol. Sci. 7:179-194, 1954.

 

Axelrod, J.: Metabolism of epinephrine and other sympathomimetic amines.
Physiol. Rev. 39:751-776, 1959.

 

Axelrod, J. and H. Weissbach: Purification and properties of hydroxy-
indole-O-methyltransferase. 1, Biol. Chem. 236:211-213, 1961.

 

Axelrod, J.: Purification and properties of phenylethanolamine-N-
methyl-transferase. g, Biol. Chem. 237:1657-1660, 1962.

 

Baldessarini, R.J. and M. Vogt: Uptake and release of NE by rat brain
tissue fractions prepared by ultrafiltration. '1, Neurochem. 18:
2519, 1971.

139

Baldessarini, R.J. and I.J. Kopin: The effect of drugs on the release
of norepinephrine-H from central nervous system tissue by electri-
cal stimulation 12_vitro. g, Pharmacol. Exp. Ther. 156:31-38, 1967.

Bapna, J., N.H. Neff and E.A. Costa: A method for studying norepine-
phrine and serotonin metabolism in small regions of rat brain:
Effects of ovariectomy on amine metabolism in anterior and posterior
hypothalamus. Endocrinol. 89:1345-1349, 1971.

 

Bargmann, W.: "Das Zwischenhirn-Hypqphysensystem." Berlin: Spring,
1954.

 

Barraclough, C.A.: Discussion of "The preoptic area of the brain and its
relation to ovulation". In: Control 21_Ovulation, edited by C.A.
Villee, New York: Pergamon Press, 1961.

Barraclough, C.A.: Modifications in the CNS regulation of reproduction
after exposure of prepubertal rats to steroid hormones. Recent
Progress 12 Hormone Res. 22:503, 1966.

Barraclough, C.A., R. Collu, R. Massa and L. Martini: Temporal inter-
relationships between plasma LH, ovarian secretion rates and peri- ‘
pheral plasma progestin concentrations in the rat: Effects of nembu-
tal and exogenous gonadotropins. Endocrino19gy 88:1437-1447, 1971.

 

Barry, J., M.P. Dubois and B. Carette: Immunofluorescence study of the
preoptico-infundibular LRF neurosecretory pathway in the normal,
castrated or testosterone treated male guinea pig. Endocrinology
95:1416-1423, 1974.

 

Bauer, K.: Degradation of thyrotropin releasing hormone (TRH). Its inhi-
bition by pryo glu-His-OCH3 and the effect of the inhibitor in
attempts to study the biosynthesis of TRH. In: Lipmann Symposium:
Enepgy, Biosynthesis and Regulation 1g_Molecular Biology, p. 53-62.
Berlin/New York: deGruyter, 1974.

 

 

 

Ben-Jonathan, N. and J.C. Porter: A sensitive radioenzymatic assay for
dopamine, norepinephrine and epinephrine in plasma and tissue.
Endocrinology 98:1497-1507, 1976.

 

Ben-Jonathan, N., R.S. Mical and J.G. Porter: Dopamine (DA) and norepi-
nephrine and/or epinephrine (NE-E) in hypophseal portal plasma,
arterial plasma, and hypothalamic tissue. The Endocrine Society,
57th Ann. Meeting Abst. 291, pp. 196, 1975.

Benedetti, L.W., R. Lozdziejsky, M.S. Sala, J.M. Monti and E. Gimo:
Blockade of ovulation after atropine implants in the lateral hypo-
thalamus of the rat. Experientia 27:1159-1160, 1971.

Benoit, J. and I. Assenmacher: The control by visible radiations of the
gonadotropic activity of the duck hypophysis. Recent Prggress 1n_
Hormone Res. 15:143-164, 1959.

 

 

140

Benoit, J. and I. Assenmacher: Recherches sur la photosensibilité des
recepteurs nervewx superficiel et profond dans la gonadostimulation
per les radiations visibles chez le canard Pekin impubere. Compt.
Rend. Acad. Sci. 262:2750-2752, 1966.

 

Bergland, R.M. and R.B. Page: Can the pituitary secrete directly to the
brain? (Affirmative Anatomical Evidence.) Endocrinology 102:1325-
1338, 1978.

 

Beyer, C. and C.H. Sawyer: In Frontiers 1E_Neuroendocrinology 1969,
edited by W.E. Ganong and L. Martini, p. 255. New York/London/
Toronto: Oxford University Press, 1969.

 

Bingel, A.S. and N.B. Schwartz: Pituitary LH content and reproductive
tract changes during the mouse oestrous cycle. 1, Rgprod. Fertility
19:215-222, 1969.

 

Bishop, W., C.P. Fawcett, C. Krulich and S.M. McCann: Acute and chronic
effects of hypothalamic lesions on the release of FSH, LH and pro-
lactin in intact and castrated rats. Endocrinolggy 91:643-656,
1972.

 

Bissonnette, T.H.: Modification of mammalian sexual cycles: Reaction
of ferrets (Putorius vulgaris) of both sexes to electric light
added after dark in November and December. Proc. 39y. Soc. London,
§gr, B 110:322-336, 1932.

 

 

Bissonnette, T.H.: Modification of mammalian sexual cycles. V. The
avenue of reception of sexually stimulating light. 1, Comp.
Psychol. 22:93-103, 1936.

Bissonnette, T.H.: Influence of light on the hypophysis. Effects of
long-continued "night lighting" on hypophysectomized female ferrets
and those with optic nerves cut. Endocrinolpgy 22:92, 1938.

Bissonnette, T.H.: Experimental modification of breeding cycles in
goats. Physiol. 2001. 14:379-383, 1941.

 

Bissonnette, T.H. and A.G. Csech: Modification of mammalian sexual
cycles. VII. Fertile matings of raccoons in December instead of
February induced by increasing daily periods of light. Proc. 32y.
Soc. London, Ser. §_122:246-254, 1937. ~

 

Bissonnette, T.H. and A.P.R. Wadlund: Duration of testis activity of
Sturnus vulgaris in relation type of illumination. ‘1, Expt. Biol.
9:339-350.

 

Blake, C.A.: Effects of pinealectomy on rat pituitary gonadotropin
release. Fed. Proc. 34:220, Abstr. No. 10, 1975.

Blake, C.A.: A detailed characterization of the proestrous luteinizing
hormone surge. Endocrinology 98:445-450, 1976.

141

Blake, C.A. and C.H. Sawyer: Effects of hypothalamic deafferentation on
the pulsatile rhythm in plasma concentrations of luteinizing
hormone in ovariectomized rats. Endocrinolggy 94:730-736, 1974.

 

Blake, C.A., R.I. Weiner, R.A. Gorski and C.H. Sawyer: Secretion of
pituitary luteinizing hormone and follicle stimulating hormone
in female rats made persistently estrus or diestrus by hypothala-
mic deafferentation. Endocrinology 90:855-861, 1972.

 

Bliss, E.L., A. Frischot and L. Samuels: Brain and testicular function.
Life Sci. 11:231-238, 1972.

Bliss, E.L., W. Thatcher and J. Ailion: Relationship of stress to
brain serotonin and 5-hydroxyindoleacetic acid. 1, Psychiat. Res.
9:71-80, 1972.

 

Boccabella, A.V., E.D. Salgado and E.A. Alger: Testicular function and
histology following serotonin administration. Endocrinology
71:827, 1962. -

 

Boden, G., L.E. Lundy and O.E. Owen: Influence of Levodopa on serum
levels of anterior pituitary hormones in man. Neuroendocrinology
10:309-315, 1972. ~

Boling, J.L. and R.J. Blandau: The estrogen-progesterone induction of
mating responses in the spayed female rat. Endocrinology 25:359,
1939.

 

Bogdanove, E.M.: Direct gonad-pituitary feedback: An analysis of
effects of intracranial estrogenic depots on gonadotrophin secre-
tion. Endocrinology 73:696-712, 1963.

 

Bogdanove, E.M.: The role of the brain in regulation of pituitary gona-
dotropin secretion. Vitamins and Hormones 22:205-260, 1964.

 

Borgeat, P., F. Labrie, J. Drouin and A. Belanger: Inhibition of
adenosine 3',5' monophosphate accumulated in anterior pituitary
gland 12_vitro by growth hormone release-inhibiting hormone.
Biochem. Biophys. Res. Comm. 56:1052-1058, 1974.

 

 

Brodie, B.B. and P.A. Shore: Interaction of drugs with norepinephrine
in the brain. Pharmacol. Rev. 548-564, 1959.

 

Brodish, A. and C.N.H. Long: ACTH-releasing hypothalamic neurohumor
in peripheral blood. Endocrinolggy 71:298-306, 1962.

 

Browman, L.G.: Light in its relation to activity and estrous rhythms
in the albino rat. '1, Exp. 2001. 75:375-388, 1937.

Brown, P.S.: The effect of 5-HT and other drugs on induced ovulation
in immature mice. g, Endocrinol. 35:161, 1966.

 

142

Brown, P.S.: Antigonadotrophic effects of a-methyltyrosine, methylser-
gide and reserpine. Nature 214:1268—1269, 1967.

Brown, P.S.: Pituitary follicle-stimulating hormone in immature female
rats treated with drugs that inhibit the synthesis or antagonize
the action of catecholamines and 5-hydroxytryptamine. Neuroendo-
crinology 7:183-192, 1971.

Brown, P.S. and L. Fawke: Effect of reserpine, pCPA, a-methyltyrosine,
thymoxamine or methallibure on pituitary FSH in male rats. .1.
Reprod. Fertility 28:167, 1972.

Brown-Grant, K.: The induction of ovulation by ovarian steroids in the
adult rat. g, Endocrinol. 43:553-562, 1969.

Brown-Grant, K.: In: Steroid Hormones and Brain Function, edited by
C.H. Sawyer and R.A. Gorski, UCLA Forum Med. Sci. No. 15, p. 269.
Los Angeles: Univ. of California Press, 1974.

Brown-Grant, K.: Steroid hormone administration and gonadotrophin secre-
tion in the gonadectomized rat. g, Endocrinol. 62: 319-332, 1974.

 

Brown-Grant, K., J.M. Davidson and F. Grieg: Induced ovulation in
albino rats exposed to constant light. 1, Endocrinol. 57:7-22, 1973.

 

Brown-Grant, K. and F. Naftolin: Facilitation of luteinizing hormone
secretion in the female rat by progesterone. 1, Endocrinol. 53:
37-46, 1972.

 

Bunn, J.F., J.W. Everett: Ovulation in persistent-estrous rats after
electrical stimulation of the brain. Proc. Soc. Exp. Biol. Med.
96:369-371, 1957.

 

 

Burgus, R., T.F. Dunn, D. Desiderio and R. Guillemin: Structure
moléculaire du facteur hypothalamique hypophysiotrope TRF d'origine
ovine: Mise en evidence par spectrometrie de masse de la sequence
PCA-His-Pro-NHZ. C.R. Acad. Sci. (Paris) 269:1870-1873, 1969.

Burgus, R. and R. Guillemin: Hypothalamic releasing factors. Agg-
Rev. Biochem. 39:499-526, 1970.

 

 

Burkhardt, J.: Transition from anoestrus in the mare and the effects
of artifical lighting. 1,'Ag_. Sci. 37:64-68, 1947.

Burns, J.H., D.J. Finney and L.G. Goodwin: Biological Standardization,
London: Oxford University Press, 1950.

 

Cajal, S.R.: Los fenomenos precoces de la degeneracion traumatica de
las vias centrales. Rev. Clin. g3 Madrid 5:201-206, 1911.

Caligaris, L., J.J. Astrada and S. Taleisnik: Stimulating and inhibiting
effects of progesterone on the release of luteinizing hormone.
Acta Endocr. (Kbh.) 59:177-185, 1968.

 

143

Campbell, J.H., G. Feuer, J. Carcia and G.W. Harris: The infusion of
brain extracts into the anterior pituitary gland and the secretion
of gonadotrophic hormone. g, Physiol. (London) 157:30P-31P, 1961.

Campbell, 6.8. and N.B. Schwartz: Timing of events of the second estrous
cycle in constant light (LL). Proc. 21_the 7th Annual Meetipg 91
the Society for the Study 2: Reproduction, p. 120, Abstr. No. 116,
1974.

 

 

Campbell, 0.8., N.B. Schwartz and M.G. Firlit: The role of adrenal and
ovarian steroids in the control of serum LH and FSH. Endocri-
nology 101:162, 1977.

Canivenc, R. and M. Bonnin-Laffargue: Inventory of problems raised by
the delayed ova implantation in the European badger (Meles meles
L.). In: Delayed Implantation, edited by A.G. Enders. Chicago:
Univ. of Chicago Press, p. 115-128, 1963.

 

 

Carlsson, A., B. Falck and N.A. Hillarp: Cellular localization of brain
monoamines. Acta Physiol. Scand. 56, Suppl. 196, 1-28, 1962.

 

Carlyle, A. and S.B. Carter: The influence of light on the occurrence
of ovulation in the rat. 1, Physiol. (London) 157:44p, 1961.

Carrer, H.F. and S. Taleisnik: Neural pathways associated with mesen-

cephalic inhibitory influence on gonadotropin secretion. Brain
Res. 38:299-313, 1972.

Carrer, H.F. and S. Taleisnik: Effect of mesencephalic stimulation on
the release of gonadotropins. J, Endocrinol. 48:527-539, 1970.

 

Carter, S.B.: The relationship of the time of ovulation to the activity
pattern in the rat. J, Endocrinol. 53:333-334, 1972.

 

Chappel, 8.0. and C.A. Barraclough: Hypothalamic regulation of pitui-
tary FSH secretion. Endocrinology 98:927-935, 1976.

 

Chappel, S.C. and C.A. Barraclough: Further studies on the regulation
of FSH secretion. Endocrinology 101:24-31, 1977.

 

Chen, C.L. and J. Meites: Effects of estrogen and progesterone on serum
and pituitary prolactin levels in ovariectomized rats. Endocri-
nology 86:503-505, 1970.

Chen, C.L., Y. Amenomori, K.H. Lu, J.L. Voogt and J. Meites: Serum pro-
lactin levels in rats with pituitary transplants or hypothalamic
lesions. Neuroendocrinolggy 6:220-227, 1970.

 

Cheng, C.P., G. Sayers, L.S. Goodman and C.A. Swinyard: Discharge of
ACTH from transplanted pituitary tissue. Ag. :1. Physiol. 159:
426-432, 1949.

144

Choudhury, S.A.R., R.M. Sharpe and P.S. Brown: Pituitary FSH activity
in rats treated with apomorphine, pimozide and drugs that modify
catecholamine levels. Neuroendocrinolqu 12:272, 1973.

 

Chowers, I. and S.M. McCann: Content of luteinizing hormone releasing
factor and luteinizing hormone during the estrous cycle after
changes in gonadal steroid titers. Endocrinology 76:700-708, 1965.

 

Chowers, I. and S.M. McCann: Comparison of the effect of hypothalamic
and pituitary implants of estrogen and testosterone on reproductive
system and adrenal of female rats. Proc. Soc. Exp. Biol. Med. 124:
260-266, 1967.

 

 

Chu, R.W.: Effect of environmental illumination on oestrous cycles of
rodents. Acta Cytol. 9:221-227, 1965.

 

Ciarnello, R.D., R.B. Barchas and G.S. Byers: Enzymatic synthesis of
adrenaline in mammalian brain. Nature (London) 221:368-369, 1969.

Clarke, J.R. and J.F. Kennedy: Effect of light and temperature upon
gonad activity in the mole (Microtus agrestis). Gen. Comp.
Endocrinol. 8:474-488, 1967.

 

Clegg, M.T. and W.E. Ganong: Environmental factors affecting reproduc-
tion. In: Reproduction 1n_Domestic Animals (2nd ed.), edited by
H.H. Cole and P.T. Cupps. New York: Academic Press, p. 473-488,
1969.

 

 

Clemens, J.A., Y. Amenomori, T. Jenkins and J. Meites: Effects of hypo-
thalamic stimulation, hormones and drugs on ovarian function in old
female rats. Proc. Soc. Exp. Biol. Med. 132:561-563, 1969.

 

 

Cohen, E.L. and R.J. wurtman: Brain acetylcholine: Control by dietary
choline. Science 191:561-562, 1976.

Collu, R., F. Fraschini and L. Martini: Role of indoleamines and cate-
cholamines in the control of gonadotrophin and growth hormone
secretion. In: Drng Effects 22_Neuroendocrine Regulation (Prog.
Brain Res., Vol. 39) E. Zimmermann, W.H. Gispen, B.H. Marks and D.
DeWied (Eds.), Amsterdam: Elsevier, pp. 289-299, 1973.

 

Colombo, J.A., D.M. Baldwin and C.H. Sawyer: Timing of the estrogen-
induced release of LH in ovariectomized rats under an altered
lighting schedule. Proc. Soc. Exp. Biol. Med. 145:1123-1127, 1974.

 

Cooper, K.F., C.P. Fawcett and S.M. McCann: Variations in pituitary
responsiveness to luteinizing hormone releasing factor during
the rat oestrous cycle. g, Endocrin. 57:187-188, 1973.

Coppola, J.A.: The apparent involvement of the sympathetic nervous
system in the gonadotrophin secretion in female rats. g, Reprod.
Fertility, Suppl. 4:35-39, 1968.

145

Coppola, J.A.: Turnover of hypothalamic catecholamines during various
states of gonadotropin secretion. Neuroendocrinology_5:75-80,
1969.

 

Corbin, A. and E.L. Daniels: Changes in concentration of female rat
pituitary FSH and stalk-median eminence follicle stimulating hor-
mone releasing factor with age. Neuroendocrino1ggy 2:304-314, 1967.

 

Corbin, A. and E.L. Daniels: Induction of puberty in immature female
rat: Effect of estrogen on pituitary FSH and stalk-median-eminence
FSH-releasing factor. Neuroendocrinolpgy 4:65-74, 1969.

 

Corbin, A. and E.A. Schottelius: Hypothalamic neurohormonal agents and

sexual maturation of immature female rats. ‘ém, g, Physiol. 201:
1176-1180, 1961.

Corbin, A. and G. Upton: Effect of synthetic luteinizing hormone re-
leasing hormone on pituitary and serum levels of luteinizing hor-
mone of intact and median eminence lesioned male rats. Experientia
29:1025-1026, 1973.

 

Costa, E. and J.L. Meek: Regulation of biosynthesis of catecholamines
and serotonin in CNS. Am, R, Pharm. 14:491-511, 1974.

Costa, E. and N.H. Neff: In: Biochemistry and Pharmacolq yup: the Basal
Ganglia, edited by E. Costa, L.T. Cote and M.D. Yahr, pp. 141-155,
New York: Raven Press, 1966.

 

Coyle, J.T. and D. Henry: Catecholamines in fetal and newborn rat
brain. 1, Neurochemistry721:61-67, 1973.

 

Coyle, J.T. and S. Snyder: Catecholamine uptake by synaptosomes in
homogenates of rat brain: Stereospecificity in different areas.
1, Pharmacol. Exp. Ther. 170: 221-231, 1969.

 

Cramer, O.M. and C.A. Barraclough: Effect of electrical stimulation of
the preoptic area on plasma LH concentrations in proestrous in
rats. Endocrino1pgy_88:1175-1183, 1971.

 

Craven, R.P. and P.G. McDonald: The effects of microinjection of cate-
cholamines into the median eminence and preoptic region on the
blockade of ovulation by reserpine in the rat. 1, Reprod.
Fertility. 27:480—481, 1971.

Craven, R.P. and P.G. McDonald: The effect of intrahypothalamic infu-
sions of dopamine and noradrenaline on ovulation in reserpine-
treated female rats. J, Endocrinol. 58:319-326, 1973.

 

Critchlow, V.: Blockade of ovulation in the rat by mesencephalic
lesions. Endocrinolggy 63:596-610, 1958.

 

Critchlow, V.: Ovulation induced by hypothalamic stimulation in the
anesthetized rat. Am, J, Physiol. 195:171-174, 1958.

146

Critchlow, V.: The role of light in the neuroendocrine system. In:
Advances 1E Neuroendocrinology, Chap. 12, pp. 377-402, edited by
A.V. Nalbandov, Urbana: University of Illinois Press, 1963.

 

Crtichlow, B.V. and J. DeGroot: Experimental investigation of pathways
involved in light-induced constant estrus in the rat. Anat.
Record 136:179, 1960.

Currie, G.N., D.L. Black and D.T. Armstrong: Blockade of ovulation in
the rabbit with catecholamines and sympathomimetics. Proc. Soc.
Exp. Biol. Med. 130:598-602, 1969.

 

Cutuly, E.: AutOplastic grafting of anterior pituitary in male rats.
Anat. Record. 80:83-97, 1941.

 

Daane, T.A. and A. Parlow: Serum FSH and LH in constant-light-induced
persistent estrus: Short-term and long-term studies. Endocri-
nology 88:964-968, 1971.

Daane, T.A. and A.F. Parlow: Periovulatory patterns of rat serum follicle
stimulating hormone and luteinizing hormone during the normal estrous
cycle: Effects of pentobarbital. Endocrinology 88:653-663, 1971.

 

Dahlstrbm, A.: Naunyn-Schmiedebe§g§_Arch. Pharmakol. Exp. Pathol. 257,
93: see Barchast g£_a1, (1972). In: Hormones and Behaviour,
edited by S. Levine, p. 235. New York: Academic Press, 1967.

 

 

Dahlstr6m, A. and K. Fuxe: Evidence for the existence of monoamine-
containing neurons in the central nervous system. Acta Physiol.
Scand. 62(Supp1. 232):1, 1964.

 

Daniel, P.M.: The anatomy of the hypothalamus and pituitary gland.
In: Neuroendocrinolqu, Vol. I, edited by L. Martini and W.F.
Ganong, pp. 15-80, New York: Academic Press, 1966.

 

David-Nelson, M.A. and A. Brodish: Evidence for a diurnal rhythm of
corticotropin releasing factor (CRF) in the hypothalamus.
Endocrinology 85:861-866, 1969.

 

Davidson, J.M.: Feedback regulation of gonadotropin secretion. In:
Frontiers 13_Neuroendocrinology, edited by W.F. Ganong and L.
Martini, pp. 343-388, Vol. 1, Oxford Univ. Press, 1969.

 

Davidson, J.M., E.R. Smith and C.Y. Bowers: Effects of mating on
gonadotropin release in the female rat. Endocrinology_93:ll85-
1192, 1973.

 

Debeljuk, L., A. Arimura and A.V. Schally: Effect of estradiol and pro-
gesterone on the LH release induced by LH-releasing hormone (LH-RH)

in intact diestrous rats and anestrous ewes. Proc. Soc. Exp. Biol.
Med. 139:774-777, 1972.

147

Debeljuk, L., V.M. Feder, and O.A. Paulucci: Effect of treatment with
melatonin in the pituitary testicular axis of the male rat.
1, Reprod. Fertilipy 21:363-364, 1970.

 

Dempsey, E.W., H.J. Myers, W.C. Young and D.B. Jennison: Absence of
light and the reproductive cycle in the guinea pig. 12,.1.
Physiol. 109:307-311, 1934.

Dempsey, E.W. and H.F. Searles: Environmental modification of certain
endocrine phenomena. Endocrinology 32:119-128, 1943.

 

Dermody, W.C., E.A. Becvar, B.L. Windsor and J.R. Reel: Radioimmunoassay
of luteinizing hormone releasing factor (LRF). Fed. Proc. 32:239
(Abstract), 1973.

Deuben, R.R. and J. Meites: Stimulation of pituitary growth hormone
release by hypothalamic extract 1p_vitro. Endocrinology 74:404-
414, 1964.

 

Dewan, E.M.: On the possibility of a perfect rhythm method of birth
control by periodic light stimulation. Am, 1, Obstet. Gynecol.
99:1016-1019, 1967.

 

Dickey, R.P. and B.B. Marks: Adrenergic and cholinergic mechanisms in
the regulation of gonadotropin secretion in ovariectomized rats.
Fed. Proc. 30:253, 19

Dierschke, D.J., A.N. Bhattacharya and L.E. Atkinson: Circhoral oscilla-
tions of plasma LH levels in the ovariectomized Rhesus monkey.
Endocrinolggy 87:850-853, 1970.

 

Donoso, A.O., W. Bishop, C.P. Fawcett, L. Krulich and S.M. McCann:
Effects of drugs that modify brain monoamine concentrations on

plasma gonadotropin and prolactin levels in the rat. Endocrinology
89:774-784, 1971.

 

Donovan, B.T.: The effect of light upon reproductive mechanisms, as
illustrated by the ferret. In: The Effects 91 External Stimuli gp_
_Rgproduction, edited by G.E.W. Wolstenholme and M. O'Connor.
Boston: Little, Brown & Co., p. 43-70, 1967.

 

 

 

Donovan, B.T. and J.J. Van der Werff ten Bosch: The cervical sympathe-
tic system and light-induced oestrus in the ferret. '1, Physiol.
(London) 132:123-129, 1956.

Donovan, B.T. and J.J. Van der Werff ten Bosch: Hypothalamus and
sexual maturation in rat. ‘1, Physiol. (London) 147:78-92, 1959.

Doyle, L.L. and M.T. Clegg: Effects of progesterone withdrawal on time
of oestrus and mating behavior in female rats. 1, Reprod.
Fertility 2:143-147, 1961.

148

Dufy-Barbe, L., P. Franchimont and J.M.A. Faure: Time-courses of LH and
FSH release after mating in the female rabbit. Endocino1ggy
92:1318—1321, 1973.

 

Dziuk, P.J. and B. Cook: Passage of steroids through silicone rubber.
Endocrinology 78:208-211, 1966.

 

Eik-Nes, R.B. and K.R. Brizzee: Some aspects of corticotrophin secre-
tion in the trained dog. I. The presence of a corticotrophin re-
leasing factor in the blood stream of dogs shortly after hypophy—
sectomy. Acta Endocr. (Kbh.) 29:219-223, 1958.

 

Ekins, R.P.: Theoretical aspects of saturation analysis; in Dicsfalusy
Steroid assay by protein binding. 2nd Karolinska symp osium, pp.
9-30 (Bogtrykkeiet Forum, Copenhagen), 1970.

Ely, C.A. and W.B. Schwartz: Elucidation of the role of the luteinizing
hormone in estrogen secretion and ovulation by use of an anti-
gonadotropic sera. Endocrinolggy 89:1103-1108, 1971.

 

Enders, R.K. and A.G. Enders: Morphology of the female reproductive
tract during delayed implantation in the mink. In: Delayed
Implantation, edited by A.G. Enders. Chicago: Univ. of Chicago
Press, p. 129-139, 1963.

 

Endersby, C.H., J.N. Robson, F.M. Sullivan and G.A. Wilson: Effect of
S-hydroxytryptamine on ovulation in rats. 1, Endocrinol. 48:13,
1970.

 

Endersby, C.H. and C.A. Wilson: Effect of ovarian steroids on uptake
of H—3-noradrenaline, H-3-dopamine, and H-3-5-hydroxytryptamine
by hypothalamic tissue 12_vitro. BE, 1, Pharmacol. 47:647-648, 1973.

Erdheim, J. and E. Stumme: Ueber die Schwangerschaftsveranderung der
Hypophyse. Beitr. Pathol. Anat. Allgem. Pathol. 46:1, 1909.

 

 

 

Evans, R.M.: Clinical manifestations of dysfunction of the anterior
pituitary. In: Glandular Physiology and Therapy, American Medical
Association. Chicago, p. 7, 1935.

 

Evans, J.S. and M.B. Nikitovitch-Winer: Functional reactivation and
cytological restoration of pituitary grafts by continuous local
intravascular infusion of median eminence extracts. Neuroendo-
crinology 4:83-100, 1969.

Everett, J.W.: Spontaneous persistent estrus in a strain of albino
rats. Endocrinology 25:123-127, 1939.

 

Everett, J.W.: Restoration of cyclic estrous and ovulation in persistent
estrus rats by progesterone. Anat. Record 76(Suppl.):21, 1940.

 

Everett, J.W.: Certain functional interrelationships between sponta-
neous persistent estrus, "light estrus" and short day anestrus in
the albino rat. Anat. Record 82:409, 1942.

 

149

Everett, J.W.: Progesterone and estrogen in the experimental control
of ovulation time and other features of the estrous cycle in the
rat. Endocrinology 43:389-405, 1948.

 

Everett, J.W.: Factors affecting the success of pharmacological block-
ade of ovulation in rats. 1, Clin. Endocrinol. 12:923, 1952.

 

Everett, J.W.: The mammalian female reproductive cycle and its con-
trolling mechanisms. In: Sex and Internal Secretions (3rd ed.),
edited by W.C. Young. Baltimore: Williams and Wilkins, Vol. I,
p. 497-555, 1961.

 

Everett, J.W.: Photoregulation of the ovarian cycle in the rat. In:
La Photorégulation 13.1E_Rgproduction chez les Oiseaux g£_Mammi-
feres. Colloques Internationaux du Centre National de la Recherche
Scientifique, No. 172, pp. 387-408, 1970.

 

 

 

Everett, J.W. and R.M. Radford: Irritative deposits from stainless steel
electrodes in the preoptic rat brain causing release of pituitary
gonadotropin. Proc. Soc. Exp. Biol. 108:604-609, 1961.

 

Everett, J.W. and C.H. Sawyer: A neural timing factor in the mechanism
by which progesterone advances ovulation in the cyclic rat.
Endocrinology 45:581-595, 1949.

 

Everett, J.W., C.H. Sawyer and J.E. Markee: A neurogenic timing factor
in control of the ovulating discharge of luteinizing hormone in
the cyclic rat. Endicrinology_44:234-250. 1949.

 

Everett, J.W. and C.H. Sawyer: A 24 hour periodicity in the LH releasing
apparatus of female rats, disclosed by barbiturate sedation.
Endocrinology 47:198-218, 1950.

 

Everett, J.W. and T. Tejasen: Time factor in ovulation blockade in
rats under differing lighting conditions. Endocrinology_80:790-
792, 1967.

 

Fajer, A.B., D. Hoffman and E. Shillito: Inhibitory effect of para-
chlorophenylalanine on the sexual maturation of female rats. 1,
Reprod. Fertility 22:379-380, 1970.

 

Farnebo, L.0., B. Hamberger and G. Jonsson: Release of 3-H-noradrenaline
and 3-H-dopamine from field stimulated cerebral cortex slices.
Effect of tyrosine hydroxylase and dopamine hydroxylase inhibition.
1, Neurochem. 18:2491-2500, 1971.

Farner, D.S.: Comparative physiology: Photoperiodicity. Ann. Rev.
Physiol. 23:71-96, 1961.

Fawke, L. and P.S. Brown: Pituitary content of follicle-stimulating
hormone in the female rat. .1, Reprod. Fertility 21:303-312, 1970.

150

Ferin, M., A. Tempone, P. Zimmering and R.L. Vande Wiele: Effect of
antibodies to 17 B-estradiol and progesterone on the estrous cycle
of the rat. Endocrinology_85:1070-1078, 1969.

 

Ferin, M., I. Dyrenfurth, S. Cowchock, M. Warren and R.L. Vande Wiele:
Active immunization to 17 B-estradiol and its effects upon the

reproductive cycle of the rhesus monkey. Endocrinology 94:765—
776, 1974.

 

Feder, H.H., K. Brown-Grant and G.S. Corker: Pre-ovulatory progesterone,
the adrenal cortex and the 'critical period' for luteinizing hormone
release in rats. 1, Endocrinol. 50:29-39, 1971.

 

Feder, H.H., J.A. Resko and R.W. Goy: Progesterone levels in the adrenal
plasma of preovulatory and ovariectomized rats. 1, Endocrinol. 41:
563-564, 1968.

 

Fink, 6.: Nature of luteinizing hormone releasing factor in hypophysial
porta1 blood. Nature (London) 215:159-161, 1967.

Fink, G.: The responsiveness of the anterior pituitary gland to luteini-
zing hormone releasing factor in rats exposed to constant light.
1, Endocrinol. 65:439-445, 1975.

 

Fink, G. and M.S. Aiyer: Gonadotrophin secretion after electrical stimu-
lation of the preoptic area during the oestrous cycle of the rat.
1, Endocrinol. 62: 589-604, 1974.

 

Fink, G., F. Greig, S. Chiappa and S.R. Henderson: Peripheral plasma
luteinizing hormone releasing factor and the spontaneous and reflex
release of luteinizing hormone in the rat. 1, Endocrinol. 63:33P—
34P, 1974.

 

Fink, G. and G.W. Harris: The luteinizing hormone releasing activity of
extracts of blood from the hypophysial porta1 vessels of rats.
1, Physiol. (London) 208:221—241, 1970.

Fink, G., R. Nallar and W.C. WOrthington, Jr.: Determination of luteini-
zing hormone releasing factor (LRF) in hypophysial porta1 blood.
1, Physiol. (London) 183:20P-21P, 1966.

Fink, G., R. Nallar and W.C. Worthington, Jr.: The demonstration of
luteinizing hormone releasing factor in hypophysial porta1 blood of
pre-oestrous and hypophysectomized rats. 1, Physiol. (London) 191:
407-416, 1967.

Finney, D.J.: Statistical Method 12_Biolggical Assay (2nd ed.).
London: Charles Griffin, 1964.

 

Fiske, V.M.: Effect of light on sexual maturation, estrous cycles and
anterior pituitary of the rat. Endocrinology_29:187-196, 1941.

 

151

Fiske, V.M.: Effects of light and darkness on activity of the pituitary
of the rat. Proc. Soc. Exp. Biol. Med. 40:189-191, 1939.

Fiske, V.M. and R.0. Creep: Neurosecretory activity of rats under con-
ditions of continuous light or darkness. Endocrinology 64:175-
185, 1959.

 

Flerko, B. and V. Bordos: Pituitary hypertrophy after anterior hypo-
thalamic lesions. Acta Endocrinol. (Kbh.) 35:375-380, 1960.

 

Fortier, 0.: Dual control of adrenocorticotropin release. Endocrinology
49:782-788, 1951.

Fraschini, F.: Role of indolamines in the control of the secretion of
pituitary gonadotropins. In: Neurochemical Aspect gf_Hypothalamic
Function, edited by L. Martini and J. Meites. New York: Academic
Press, pp. 141-159, 1970.

 

Fraschini, F., R. Collu and L. Martini: Mechanisms of inhibitory action
of pineal principles on gonadotropin secretion. In: The Pineal
Gland, edited by G.E.W. Wolstenholme and J. Knight. London:
Churchill, p. 259-273, 1971.

 

Freeman, M.E., R.L. Butcher and N.W. Fugo: Alteration of oocytes and
follicles by delayed ovulation. Biology 2£_Reproduction 2:209-215,
1970.

Freeman, M.E., L.E. Reichert and J.D. Neill: Regulation of the proestrous
surge of prolactin secretion by gonadotropin and estrogens in the
rat. Endocrinology 90:232—238, 1972.

 

Friedgood, B.B.: The nervous control of the anterior hypothysis (origi-
nally presented at the Harvard Tercentenary Celebrations, Sept. 15,
1936). 1, Reprod. Fertility (Suppl.) 10:3, 1970.

 

Fuxe, K.: Cellular localization of monoamines in the median eminence and
infundibular stem of some mammals. Acta Physiol. Scand. 58:383-384,
1963.

Fuxe, K.: Evidence for the existence of monoamine neurons in the central
nervous system. IV. The distribution of monoamine nerve terminals
in the central nervous system. Acta Physiol. Scand. (Suppl. 247)
64:39, 1965.

Fuxe, K., T. Hokfelt and M. Ritzen: Studies on uptake of intraventricu-
larly administered tritiated noradrenaline and 5-hydroxytryptamine
with combined fluorescence, histochemical and autoradiographic
techniques. Histochemie 16:186-194, 1968a.

Fuxe, K., T. Hfikfelt and U. Ungerstedt: Localization of indole-alkyl-
amines in CNS. Adv. Pharmacol. 6A:235-251, 1968b.

 

152

Fuxe, K. and T. Hokfelt: Catecholamines in the hypothalamus and the pi-
tuitary gland. In: Frontiers in Neuroendocrinology, edited by
W. F. Ganong and L. Martini, Oxford University Press, pp. 47-96,1969.

 

Fuxe, K. and T. Hokfelt: Participation of central monoamine neurons in
the regulation of anterior pituitary function with special regard
to the neuroendocrine role of tuberoinfundibular dopamine neurons.
In: Aspects 21_Neuroendocrinology, edited by W. Bargmann and H.
Scharrer. Berlin: Springer-Verlag, pp. 275-314, 1970.

 

Ganong, W.F., M.D. Shepherd, J.R. Wall, E.E. Van Brunt and M.T. Clegg:
Penetration of light into the brain of mammals. Endocrinology
72:962-963, 1963.

 

Gay, V.L. and A.R. Midgley, Jr.: Response of the adult rat to orchidec—
tomy and ovariectomy as determined by LH radioimmunoassay. Endo-
crinology 84:1359-1364, 1969.

Gay, V.L. and N.A. Sheth: Evidence for a periodic release of LH in
castrated male and female rats. Endocrinology 90:158-162, 1972.

 

Gazire, L.C. and W. Ladosky: Monoamine oxidase variation during sexual
differentiation. Neuroendocrinology 12:249-256, 1973.

 

Gerich, J.E., R. Lovinger and G.M. Grodsky: Inhibition by somatostatin
of glucagen and insulin release from the perfused rat pancreas in
response to arginine, iSOproterenol, and theophylline. Evidence

for a preferential effect on glucagen secretion. Endocrinology
96:749-754, 1975.

 

Gibori, G., E. Antczak and I. Rothchild: The role of estrogen in the
regulation of luteal progesterone secretion in the rat after day
12 of pregnancy. Endocrinology 100:1483, 1977.

 

Glowinski, J. and J. Axelrod: Effects of drugs on the uptake, release,
and metabolism of H3-norepinephrine in the rat brain. 1, Pharmacol.
Exp. Ther. 149:43-49, 1966.

Glowinski, J. and L.L. Iversen: Regional studies of catecholamine in
the rat brain. I. The disposition of H3—norepinephrine, H3-dopamine,
and H3-Dopa in various regions of the brain. 1, Neurochem. 13:
655-669, 1966.

Glowinski, J., I. Kopin and J. Axelrod: Metabolism of H3-norepinephrine
in the rat brain. 1, Neurochem. 12:25-30, 1965.

Goldberg, R. C. and E. Knobil: Structure and function of intraocular
hypophysial grafts in the hypophysectomized male rat. Endocri-
nology 61: 742- 752, 1957.

Goodwin, J.S., R.I. Katy, and I.J. Kopin: Effect of bromide on evoked
release of monoamines from brain slices and intact atria.
Nature (London) 221:556-557, 1969.

153

Gordon, J.R. and S. Reichlin: Changes in pituitary responsiveness to
luteinizing hormone-releasing factor during the rat estrous cycle.
Endocrinology 94:9749 Y8, 1974.

 

Gorski, R.A.: Localization and sexual differentiation of the nervous
structures which regulate ovulation. .1, Reprod. Fertility
(Suppl.) 1:67-88, 1966.

 

Gorski, R.A. and J.W. Wagner: Gonadal activity and sexual differentia-
tion of the hypothalamus. Endocrinology 76:226-239, 1965.

 

Grahame-Smith, D.G.: Studies 1§_vivo on the relationship between brain
tryptophan, brain S-HT synthesis and hyperactivity in rats treated
with a monoamine oxidase inhibitor and L-tryptophan. 1, Neurochem.
18:1053-1066, 1971.

Green, J.D.: Vessels and nerves of amphibian hypophyses. A stuy of the
living circulation and of the histology of the hypophysial vessel
and nerves. Anat. Record 99:21-53, 1947.

 

Green, J.D.: The comparative anatomy o f the hypOphysis, with special
reference to its blood supply and innervation. 1m, 1, Anat. 88:
225-311, 1951.

Green, J.D. and G.W. Harris: The neurovascular link between the neuro-
hypophysis and the adenohypophysis. 1, Endocrinol. 5:136—146, 1947.

 

Greenwald, G.S.: Failure of continuous light to induce constant oestrus
in the hamster. 1, Endocrinol. 28:123—124, 1963.

 

Guilian, D., L.A. Pohorecky and B.S. McEwen: Effects of gonadal steroids
upon brain S-hydroxytryptamine levels in the neonatal rat.
Endocrinology 93:1329-1335, 1973.

 

Guillemin, R., R. Burgus and W. Vale: The hypothalamic hypophysiotropic
thyrotropin releasing factor. Vitamins and Hormones 29:1, 1971.

 

Guillemin, R. and B. Rosenberg: Humoral hypothalamic control of anterior
pituitary: A study with combined tissue cultures. Endocrinology
57:599-607, 1955.

 

Guillemin, R., E. Yamazaki, M. Justiz and E. Sakiz: Présence dans un
extrait de tissus hypothalamiques d'une substance stimulant 1a
sécrétion de l'hormonehypophysaire thyréotrope (TSH). Premiere
purification par filtration sur le Sephadex. C.R. Acad. Sci. (Paris)
255:1018-1020, 1962.

 

Hafez, E.S.E.: Inhibitory action of artificial light on the sexual
season of the ewe. Nature 168:336, 1951.

154

Halasz, B.: The endocrine effects of isolation of the hypothalamus from
the rest of the brain. In: Frontiers 1n_Neuroendocrinology, edited
by W.F. Ganong and L. Martini. New York: Oxford University Press,
p. 307~342, 1969.

 

Halasz, B. and R.A. Gorski: GonadotrOphic hormone secretion in female
rats after partial or total interruption of neural afferents to the
medial basal hypothalamus. Endocrinology_80:608-622, 1967.

 

Halasz, B. and L. Pupp: Hormone secretion of the anterior pituitary
gland after physical interruption of all nervous pathways to the
hypophysiotrophic area. Endocrinology 77:553-562, 1965.

 

Halasz B., L. Pupp and S. Uhlarik: Hypophysiotrophic area in the hypo-
thalamus. 1, Endocrinol. 25:147-154, 1962.

 

Hall, R., G.M. Besser, A.V. Schally, D.B. Coy, D. Evered and D.S. Goldie:
Action of growth hormone release inhibitory hormone in healthy men
and acromegaly. Lancet 2:581-584, 1973.

Hamon, M., F. Javoy and C. Kordon: Synthesis and release of serotonin
in the median eminence of the rat. Life Sci. 9:167-173, 1970.

Hammond, J., Jr.: Failure of progesterone treatment to affect delayed
implantation in mink. ‘1, Endocrinol. 7:330-334, 1951.

 

Hammond, J., Jr.: Control by light of reproduction in ferrets and mink.
Nature 167:150-151, 1951.

Hansen, A.P., H. Oeskov, K. Sayer-Hansen and K. Lundback: Some actions
of growth hormone release inhibiting factor. 1;, Med. 1, 2:523-
524, 1973.

Hardin, G.M.: Sex differences and the effects of testosterone injections
on biogenic amine levels of neonatal rat brain. Brain Res. 59:
437-439, 1973.

Hardy, D.F.: The effect of constant light on the estrous cycle and
behavior of the female rat. Physiol. Behav. 5:421-425, 1970.

 

Harris, G.W.: Electrical stimulation of hypothalamus and mechanism of
neural control of adenohypophysis. 1, Physiol. (London) 107:418,
1948.

Harris, G.W.: Neural control of the pituitary gland. Monographs g:
the Physiology_Society, Arnold, London, 1955.

 

 

Harris, G.W. and D. Jacobsohn: Functional grafts of anterior pituitary
gland. Proc. Roy. Soc. Ser. B 139:263, 1952.

 

 

Harris, G.W.: Central control of pituitary secretion. In: Handbook 2:
Physiology (Am. Physiol. Soc., edited by J. Field), Section 1, Vol.
2, pp. 1007-1038 Baltimore: Williams and Wilkins, 1960.

155

Harris, G.W.: Unsolved problems in the portal vessel-chemotransmitter
hypothesis. In: ‘Hypophysiotrophic Hormones o£_the Hypothalamus,
edited by J. Meites, Proc. of the Workshop Conference, Tucson,
Arizona. Baltimore: Williams and Wilkins, p. l, 1969.

Harris, G.W. and D. Jacobsohn: Functional grafts of the anterior
pituitary gland. Proc. Roy. Soc. Ser. 1 139:263-276, 1952.

 

Harris, G.W., and K.B. Ruf: Luteinizing hormone releasing factor in rat
hypophysial portal blood collected during electrical stimulation of
hypothalamus. 1, Physiol. (London) 208:243-250, 1970.

Hayhow, W.R., C. Webb and A. Jervie: The accessory optic fiber system
in the rat. .1. Comp. Neurol. 115:187-216, 1960.

 

Heimer, L. and W.J.H. Nauta: The hypothalamic distribution of the stria
terminalis in the rat. Brain Res. 13:284-297, 1969.

Hemmingsen, A.M. and N.B. Krarup: Rhythmic diurnal variation in the
oestrous phenomena of the rat and their susceptibility to light
and dark. Biol. Med. 1, 13:1-61, 1937.

Herbert, J.: The pineal gland and light-induced oestrus in ferrets.
1, Endocrinol. 43:625-636, 1969.

 

Hiroshige, T., M. Sakakura and S. Itoh: Diurnal variation of cortico-
tropin-releasing activity in the rat hypothalamus. Endocr. Jap.
16:465-467, 1969.

 

Hiroshige, T., T. Sato, R. Ohta and S. Itoh: Increase of corticotropin-
releasing activity in the rat hypothalamus following noxious
stimuli. Jap. 1, Physiol. 19:866-875, 1969.

Hidaka, H., T. Nagatsu and K. Yagi: Occurrence of a serotonin sulpho-
transferase in the brain. '1, Neurochem. 76:783-785, 1969.

Himwich, H.E.: In: Biochemistgy, Schizophrenias and the Affective
Illnesses. Baltimore: Williams and Wilkins, 1971.

Hoffmann, J.C.: Effects of light deprivation on the rat oestrous cycle.
Neuroendocrino1ogy'2:1—10, 1967.

 

Hoffmann, J.C.: Effect of photoperiod on estrous cycle length in the
rat. Endocrinology 83:1355-1357, 1968.

 

Hoffmann, J.C.: Light and reproduction in the rat: Effect of lighting
schedule on ovulation blockade. Biol. Reprod. 1:185-188, 1969.

Hoffmann, J.C.: Light and reproduction in the rat: Effects of photo-
period length on albino rats from two different breeders. Biol.

156

Hoffmann, J.C.: The influence of photoperiods on reproductive functions
in female mammals; in Greep and Astwood Handbook of Physiology,
Section 7, Endocrinology, Vol. II, part 1, pp. 57-77 (Am. Physiol.
Soc., Bethesda), 1973.

 

Hoffmann, J.C. and A. Cullin: Effects of pinealectomy and constant

light on the estrous cycles of female rats. Neuroendocrinology
16:167—174, 1975.

 

Hoffmann, J.C. and A. Cullin: Effect of photoperiod length on feedback

sensitivity to estradiol in the female rat. Neuroendocrinology
23:285-296, 1977.

 

Hoffmann, J.C., C. Kordon and J. Benoit: Effect of different photoperiods
and blinding on ovarian and testicular functions in normal and
testosterone-treated rats. Gen. Comp. Endocrinol. 10:109-118, 1968.

 

Hoffmann, K. and I. Kfiderling: Pinealectomy inhibits stimulation of
testicular development by long photoperiods in a hamster (Phodopus
sungorus). Experientia 31:122-123, 1975.

 

Houssay, B.A., A. Biasotti and R. Sammartino: Modifications fonction-
elles de l'hypophyse aprés les lésions infundibulatubériennes chez
1e crapaud. Compt. Rend. Soc. Biol. 120:725-726, 1935.

 

Huang, H.H., S. Marshall and J. Meites: Induction of estrous cycles in
old non-cyclic rats by prOgesterone, ACTH, ether stress, or L-dopa.
Neuroendocrinology 20:21, 1976.

 

Huang, H.H.: Relation of the neuroendocrine system to loss of repro-
ductive function in aging female rats. Ph.D. thesis, Michigan
State University, 1978.

Huang, H.H., 3. Marshall and J. Meites: Reduced positive feedback by
estrogen and prOgesterone on LH and FSH release in aging female
rats. Abstract, 62nd FASEB, 1978.

Hyppa, M. and U.K. Rinne: L—dopa, its distribution and actions on
monoamines in brain. Sci. 1. Q1. Inv. 27(Sll6):64, 1971.

Ichiyama, A., S. Nakamura and Y. Nishizuka: Enzymatic studies on the
biosynthesis of serotonin in mammalian brain. .1, Biol. Chem. 245:
1699-1709, 1970.

 

Ifft, J.D.: Evidence of gonadotropic activity of the hypothalamic
arcuate nucleus of the rat. Anat. Records 142:1-8, 1962.

 

Igarashi, M. and S.M, McCann: A hypothalamic follicle stimulating
hormone-releasing factor. Endocrinology 74:446-452, 1964.

 

Illei-Donhoffer, A., B. Flerko and B. Mess: Reduction of estradiol-
binding capacity of neural target tissues in light-sterilized rats.
Neuroendocrinology 14:187-194, 1974.

157

Iversen, L.L.: Inhibition of noradrenaline uptake by sympathomimetic
amines. 1, Pharm. Pharmacol. 16:435-437, 1965.

Iversen, L.L. and J. Glowinski: Regional studies of catecholamines in
the rat brain. II. Rate of turnover in various brain regions.
1, Neurochem. 13:671-682, 1966.

Jackson, I.M. and S. Reichlin: Thyrotropin releasing hormone (TRH)
distribution in the brain, blood and urine of the rat. Life Sci.
14:2259-2266, 1974.

Jaitley, K.D., J.M. Robson, F.M. Sullivan and C. Wilson: Inhibition of
ovulation in immature rats. 1, Endocrinol. 37:xxxi, 1968.

 

Javoy, F., V. Aged, D. Bouvet and J. Glowinski: Feedback control of
dopamine synthesis in dopaminergic terminals of the rat striatum.
1, Pharmacol. Exp. Ther. 182:454-463, 1972.

 

Jeffcoate, S.L., H.M. Fraser, A. Gunn and D.T. Holland: Radioimmuno-
assay of luteinizing hormone releasing factor. 1, Endocrinol.
57:189-190, 1973.

 

J6chle, W.: Uber den Einfluss des Lichtens auf Sexualentwicklung und
Sexualperiodik bei Saflgern. Endokrinologie 33:129-138, 1956.

 

Jonsson, G., K. Fuxe and T. H6kfe1t: On the catecholamine innervation
of the hypothalamus, with special reference to the median eminence.
Brain Res. 40:271, 1972.

Jutisz, M., A. Bérault, M.A. Novella and G. Ribot: Etude de l'action

du facteur hypothalamique LRF chez le rat 12_vivo et 12 vitro.
Acta Endocr. (Kbh.) 55:481-496, 1967.

 

Jutisz, M. and M.P. de la Llosa: Studies on the release of FSH 1§_vitro
from rat pituitary glands stimulated by hypothalamic FSH-RF.
Endocrinology 81:1193-1202, 1967.

 

Kalra, S.P.: Observations on facilitation of the preovulatory rise of
LH by estrogen. Endocrinology 96:23-28, 1975.

 

Kalra, S.P. and P.S. Kalra: Effects of circulating estradiol during rat
estrous cycle on LH release following electrochemical stimulation of
preOptic brain or administration of synthetic LRF. Endocrinology
94:845-851, 1974.

 

Kalrs, P.S., S.P. Kalra, L. Krulich, C.P. Fawcett and S.M. McCann:
Involvement of norepinephrine in transmission of the stimulatory

influence of progesterone on gonadotropin release. Endocrinology
90:1168-1176, 1972.

Kalra, P.S. and S.M. McCann: Involvement of catecholamines in feedback
mechanisms. Progr. Brain Res. 39:185-198, 1973a.

158

Kalra, S.P., K. Ajika, L. Krulich, C.P. Fawcett, M. Quijada and S.M.
McCann: Effects of hypothalamic and preoPtic electrochemical
stimulation on gonadotropin and prolactin release in proestrous
rats. Endocrinology 88:1150-1158, 1971.

 

Kalra, S.P., L. Krulich and S.M. McCann: Changes in gonadotropin-re-
leasing factor content in the rat hypothalamus following electro-
chemical stimulation of anterior hypothalamic area and during the
estrous cycle. Neuroendocrinology 12:321-333, 1973.

 

Kalra, S.P., and S.M. McCann: Variations in the release of LH in re-
sponse to electrochemical stimulation of preoptic area of medial
basal hypothalamus during the estrous cycle of the rat. Endocri-
nology 93:665-669, 1973b.

Kalra, S.P. and S.M. McCann: Effect of drugs modifying catecholamine
synthesis on LH release induced by preoptic stimulation in the rat.
Endocrinology 93:356-362, 1973c.

 

Kalra, S.P. and S.M. McCann: Effects of drugs modifying catecholamine
synthesis on plasma LH and ovulation in the rat. Neuroendocri-
nology 15:79-91, 1974.

 

Kamberi, I.A.: The role of brain monoamines and pineal indoles in the
secretion of gonadotrophins and gonadotrophin releasing factors.
Progr. Brain Res. 39:261-280, 1973.

 

Kamberi, I.A.and S.M. McCann: Effect of biogenic amines, FSH-releasing
factor (FRF) and other substances on the release of FSH by pitui-
taries incubated 12_vitro. Endocrinology 85:815-824, 1969a.

 

Kamberi, I.A. and S.M. McCann: Effect of biogenic amines and other sub-
stances on release of FSH by pituitaries incubated 13 vitro. 1,
figprod. Fertility 18:153-159, 1969b.

 

Kamberi, I.A., R.S. Mical and J.C. Porter: Luteinizing hormone releasing
activity in hypophysial stalk blood and elevation by dopamine.
Science 166:388-390, 1969.

Kamberi, I.A., R.S. Mical and J.C. Porter: ’Follicle stimulating hormone
releasing activity in hypophysial portal blood and elevation by
dopamine. Nature (London) 227:714-715, l970a.

Kamberi, I.A., R.S. Mical and J.C. Porter: Effect of anterior pituitary
perfusion and intraventricular injection of catecholamines and
indoleamines on LH release. Endocrinology 87:1-12, l970b.

 

Kamberi, I.A., H.P.G., Schneider and S.M. McCann: Action of dopamine
to induce release of FSH-releasing factor FRF from hypothalamic
tissue 12 vitro. Endocrinology 86:278, 1970.

 

159

Kamberi, I.A., R.S. Mical and J.C. Porter: Effects of melatonin and

serotonin on the release of FSH and prolactin. Endocrinology 88:
1288-1293, 1971a.

 

Kamberi, I.A., R.S. Mical and J.C. Porter: Effect of anterior pituitary
perfusion and intraventricular injection of catecholamines on FSH
release. Endocrinology 88:1003-1011, 1971b.

 

Kanematsu, S., R.J. Scaramuzzio, J. Hilliard and C.H. Sawyer: Patterns
of ovulation inducing LH release following coital, electrical, and
noradrenergic stimulation in rabbit. Biol. Reprod. 7:110, 1972.

 

Karsch, E.J., D.J. Dierschke, R.F. Weick, T. Yamaji, J. Hotchkiss and E.
Knobil: Positive and negative feedback control by estrogen of

luteinizing hormone secretion in the rhesus monkey. Endocrinology
92:799-804, 1973.

 

Kato, J.: Localization of estradiol receptors in the rat hypothalamus.
Acta Endocr. (Kbh.) 72:663-670, 1973.

 

Kato, R.: Serotonin content of rat brain in relation to sex and age.
1, Neurochem. 5:202, 1960.

Kavanagh, A. and J. Weisz: Localization of dopamine and norepinephrine
in the medial basal hypothalamus of the rat. Neuroendocrinology
13:201-212, 1973/74.

 

Kent, G.C., P.M. Ridgeway and E.F. Strobel: Continual light and con-
stant estrus in hamsters. Endocrinology 82:699-703, 1968.

 

Kincl, E.A. and H.W. Rudel: Sustained release hormonal preparations.

 

Kinson, G.A., and C.C. Liu: Long-term testicular responses to blinding
in rats. Life Sci. 14:2179-2188, 1974.

Kledzik, G.S. and J. Meites: Reinitiation of estrous cycles in light
induced constant estrous female rats by drugs. Proc. Soc. Exp.
Biol. Med. 146:989-992, 1974.

 

Knigge, K.M.: Gonadotropic activity of neonatal pituitary glands im-
planted in the rat brain. ‘ém, 1, Physiol. 202:387, 1962.

Knobil, E.: On the control of gonadotropin secretion in the rhesus
monkey. Recent Progg. Hormone Res. 30:1—46, 1974.

Kobayashi, F. and T. Miyake: Acute effect of hypothalamic deafferentation
on progesterone-induced ovulating hormone release in the rats.
Endocr. Jap. 18:395-401, 1971.

 

Kobayashi, F., K. Hara and T. Miyake: Facilitation of LH release by pro-
gesterone in proestrous rats. Endocr. Jap. 17:149, 1970.

160

Kobayashi, H. and T. Matsui: Fine structure of the median eminence and
its functional significance. In: Frontiers 12_Neuroendocrinology,
edited by W.F. Ganong and L. Martini. New York: Oxford Univer-
sity Press, pp. 3-46, 1969.

 

Koe, B.K. and A. Weisman: Marked depletion of serotonin by PCPA.
Adv. Pharmacol. 6B:29, 1968.

 

Koerker, D.J., W. Ruch, E. Chideckel, J. Palmer, C.J. Goodner, C.J.
Ensinck and 0.0. Gale: Somatostatin: Hypothalamic inhibitor of
the endocrine pancreas. Science 184:482-483, 1974.

Kordon, C.: Blockade of ovulation in the immature rat by local micro-
injection of a-methyl Dopa into the arcuate region of the hypothala-
mus. ‘1, Neurovisc. Re1.(Suppl.) 10:41, 1971.

 

Kordon, C.: Effects of selective experimental changes in regional hypo-
thalamic monoamine levels on superovulation in the immature rat.
Neuroendocrinology 4:129-138, 1969.

 

Kordon, C. and J. Glowinski: Selective inhibition of superovulation
by blockade of dopamine synthesis during the "critical period" in
the immature rat. Endocrinology 85:924-931, 1969.

 

Kordon, C. and J. Glowinski: Role of hypothalamic monoaminergic neurons
in the gonadotropin release regulating mechanism. Neuropharmacol.
11:153-162, 1972.

Kordon, C., F. Javoy, G. Vassent and J. Glowinski: Blockade of super-
ovulation in the immature rat by increased brain serotonin. Eur.

Kordon, C. and G. Vassent: Effect de microinjections intrahypothalami-
ques et intrahypophysaires k'un inhibiteur de la monoamine-oxydase
sur l'ovulation provoquée chez la ratte impubére. C.R. Acad. Sci.
(D)(Paris) 266:2473-2476, 1968.

K6ves, K. and B. Halasz: Location of the neural structures triggering
ovulation in the rat. Neuroendocrinology 6:180-193, 1970.

 

Krehbiel, R.R.: The effects of theelin on delayed implantation in the
pregnant lactating rat. Anat. Record 81:381-382, 1941.

 

Krieg, R.J. and C.H. Sawyer: Effects of intraventricular catecholamines
on luteinizing hormone release in ovariectomized steroid primed
rats. Endocrinology_99:411-419, 1976.

Krulich, L. and S.M. McCann: Evidence for the presence of growth
hormone-releasing factor in blood of hypoglycemic, hypophysectomized
rats. Proc. Soc. Exp, Biol. Med. 122:668-671, 1966.

161

Kueng, W., A. Wirz-Justice, R. Menzi and E. Chappuis-Arndt: Regional
brain variations of tryptophan, monoamines, monoamine oxidase
activity, plasma free and total tryptophan during the estrous cycle
of the rat. Neuroendocrinology 21:289-296, 1976.

 

Labhsetwar, A.P.: The role of oestrogens in spontaneous ovulation:
Evidence for positive oestrogen feedback in the 4-day oestrus cycle.
1, Endocrinol. 47:481-493, 1970.

 

Labhsetwar, A.P.: Effects of serotonin on spontaneous ovulation: A
theory for the dual hypothalamic control of ovulation. Acta
Endocrinol. (Kbh.) 68:334-344, 1971.

 

Labhsetwar, A.P. and A. Zolovick: Evidence for the theory of dual
hypothalamic control of ovulation. Nature 245:158-160, 1973.

Ladosky, W. and L.C. Gaziri: Brain serotonin and sexual differentiation
of the nervous system. Neuroendocrinology 6:168-174, 1970.

 

Lambert, H.H.: Intensity of continuous light:- Threshold lower for per-
sistent estrus than for retinal degeneration. Biol. Reprod. 13:
576-580, 1975.

 

LaMotte, R.H., D.E. Schmidt and W.S. Ruliffson: Multiple substrate
determination of monoamine oxidase distribution and iproniazid inhi-
bition in rat brain. 1, Neurochem. 16:725-730, 1969.

Lawton, I.E.: Facilitatory feedback effects of adrenal and ovarian hor-
mones on LH secretion. Endocrinology_90:575-579, 1972.

 

Lawton, I.E. and N.B. Schwartz: Pituitary LH content in rats exposed
to continuous illumination. Endocrinology 77:1140-1142, 1965.

 

Lawton, I.E. and N.B. Schwartz: Pituitary ovarian function in rats ex-
posed to constant light: A chronological study. Endocrinology
81:497-508, 1967.

 

Leavitt, W.W., C.G. Bosley and G.C. Blaha: Progestin secretion and
excretion in the ovariectomized rat bearing a luteinized ovarian
graft in the hepatic porta1 circulation. Nature 234:283, 1971.

Legan, S.J., C.A. Coon and F.J. Karsch: Role of estrogen as initiator
of daily LH surges in the ovariectomized rat. Endocrinology 96:
50-56, 1975.

 

Legan, S.J., R.L. Hauger, D.L. Foster and F.J. Karsch: A marked
seasonal variation in the mechanism controlling tonic LH secretion
in ewes. Fed. Proc. Fed. Am, Soc. Exp. Biol. 35:615, 1976.

 

 

Legan, S.J. and F.J. Karsch: A daily signal for the LH surge in the
rat. Endocrinology 96:57-62, 1975.

 

162

1e Gros Clark, W.E.: Morphological aspects of the hypothalamus. In:
The Hypothalamus, Morphological, Functional, Clinical and Surgical
Aspects, edited by W.E. 1e Gros Clark g£_§1,, Edinburgh/London:
Oliver & Boyd, pp. 1-68, 1938.

Lichtensteiger, W. and H. Langemann: Uptake of exogenous catecholamines
by monoamine containing neurons of the central nervous system:
Uptake of catecholamines by arcuate infundibular neurons. 1,
Pharmacol. Exp. Ther. 151:469-492, 1966.

 

Lippmann, W.: Relationship between hypothalamic NE and S-HT and gonado-
tropin secretion in the hamster. Nature 218:173, 1968.

Lisk, R.D. and L.R. Kannwischer: Light: Evidence for its direct effect
on hypothalamic neurons. Science 146:272-273, 1964.

Longenecker, D.E. and D.G. Gallo: Inhibition of PMSG induced ovulation
in immature rats by melatonin. Proc. Soc. Exp. Biol. Med. 137:162,
1971.

 

 

Louvet, J.F., S.M. Harman and G.T. Ross: Effects of human chorionic
gonadotropin, human interstitial cell stimulating hormone and human
follicle-stimulating hormone on ovarian weights in estrogen-primed
hypophysectomized immature female rats. Endocrinology_96:ll79-
1186, 1975.

 

Luschka, H.: Der Hirnanhang_und die Steissdrfise des Menschen. Berlin:
Reimer, 1860.

Macon, J.B., L. Sokoloff and J. Glowinski: Feedback control of rat
brain 5-hydroxytryptamine synthesis. 1, Neurochem. 18:323, 1971.

Mahesh, V.G. and B.D. Goldman: In: Proc. 3rd Int. Cong. Horm. Ster.,
edited by V.H.T. James and L. Martini, p. 662. Amsterdam:
Excerpta Medica Foundation, Int. Cong. Series No. 210, 1970.

 

 

Malamed, S., A.M. Poisner and J.M. Trifaro: The fate of the chromaffin
granule during catecholamine release from the adrenal medulla.
Biochem. Pharmacol. 17:241-246, 1968.

 

Mandell, A.J. and M. Morgan: Indole (ethyl)amine N-methyltransferase
in human brain. Nature 230:85, 1971.

Mann, D.R. and C.A. Barraclough: Role of estrogen and progesterone in
facilitating LH release in 4-day cyclic rats. Endocrinology 93:
694-699, 1973a.

 

'Mann, D.R. and C.A. Barraclough: Changes in peripheral plasma progester-
one during the rat 4-day estrous cycle: An adrenal diurnal
rhythm. Proc. Soc. 152, Biol. Med. 142:1226-1229, 1973b.

163

Mann, D.R., C.D. Korowitz, L.A. McFarland and M. Cost: Interaction of
the light-dark cycle, adrenal glands and time of steroid administra-
tion in determining the temporal sequence of LH and Prl release
in female rats. Endocrinology 99:1252-1262, 1976.

 

Marshall, F.H.A.: The breeding season; in Marshall's physiology of
reproduction, 3rd ed., vol. 1, part 1, pp. 1-42. London:
Longmans Green, 1956.

Martini, L., F. Fraschini and M. Motta: Neural control of anterior pi-
tuitary functions. Recent Progr. Hormone Res. 24:439-496, 1969.

 

McCann, S.M. and A.P.S. Dhariwal: Hypothalamic releasing factors and
the neurovascular link between the brain and the anterior pituitary.
Neuroendocrinology 1:261-297, 1966.

 

McCann, S.M., P.S. Kalra, A.O. Donoso, W. Bishop, H.P.G. Schneider, C.P.
Fawcett and L. Krulich: The role of monoamines in the control of
gonadotropin and prolactin secretion. In: Brain-Endocrine Inter-
action. Median Eminence, Structure and Function, edited by K.M.
Knigge, D.E. Scott and W. Weindl. Basel: S. Karger, pp. 224-235,
1972.

 

 

McCann, S.M. and J.C. Porter: Hypothalamic pituitary stimulating and
inhibiting hormones. Physiol. Rev. 49:240-284, 1969.

 

McCann, S.M., S. Taleisnik and R.M. Friedman: LH-releasing activity in
hypothalamic extracts. Proc. Soc. Exp. Biol. Med. 104:432-434, 1960.

 

 

McCormack, C.E.: Delayed ovulation in rats exposed to continuous light:
Influence of the level of illumination. Endocrinology 95:638-
640, 1974.

 

McCormack, C.E.: Timing of ovulation in rats eXposed to continuous
light. Fed. Proc. 35:616, 1976.

McCormack, C.E. and B. Bennin: Delay of ovulation caused by exposure to
continuous light in immature rats treated with pregnant mare's
serum gonadotropin. Endocrinology 86:611-619, 1970.

 

McCormack, C.E. and K. Sridaran: Timing of ovulation in rats during
exposure to continuous light: Evidence for a circadian rhythm
of luteinizing hormone secretion. 1, Endocrinol. 76:135-144, 1978.

 

McDermott, W., E.C. Fry, J.R. Brobeck and C.N.G. Long: Release of
adrenocorticotrOphic hormone by direct application of epinephrine
to pituitary grafts. Proc. Soc. Exp. Biol. 73:609-610, 1950.

 

McDonald, P.G. and D.P. Gilmore: The effect of ovarian steroids on
hypothalamic thresholds for ovulation in the female rat. 1,
Endocrinol. 49:421, 1971.

 

164

McGeer, E.C., S. Gibson, J.A. Wada and P.L. McGeer: Distribution of
tyrosine hydroxylase activity in adult and developing brain.
Can. 1, Biochem. 45:1943, 1967.

Meites, J., P.K. Talwalker, and A. Ratner: Evidence for a prolactin-
inhibiting factor in rat hypothalamic tissue. Program 44th
Endocrine Society, Chicago, p. 19, 1962.

 

 

Mennin, S.P., and R.A. Gorski: Effects of ovarian steroids on plasma
LH in normal and persistent estrous adult female rats. Endocri-
nology 96:486-491, 1974.

Mess, B., L. Tima and C.P. Trentini: The role of pineal principles in
ovulation. In: Drug Effects og_Neuroendocrine Regulation (Prog.
Brain Res., Vol. 39), edited by B.H. Marks and D. de Wied.
Amsterdam: Elsevier, pp. 251-273, 1973.

 

 

Meyer, D.C. and W.B. Quay: Hypothalamic and suprachiasmatic uptake of
serotonin 12_vitro: Twenty-four hour changes in male and proestrous
female rats. Endocrinology 98:1160-1165, 1976.

 

Meyer, D.C. and W.B. Quay: Effects of continuous light and darkness
and of pinealectomy, adrenalectomy and gonadectomy on uptake of H-
serotonin by the suprachiasmatic nuclear region of male rats.
Neuroendocrinology 22:231-239, 1977.

 

Mittler, J.C. and J. Meites: .22 vitro stimulation of pituitary follicle-
stimulating hormone release by hypothalamic extract. Proc. Soc.
Exp. Biol. Med. 117:309-313, 1964.

 

Mittler, J.C., T.W. Redding and A.V. Schally: Stimulation of thyrotro-
pin (TSH) secretion by TSH-releasing factor (TRF) in organ cultures
of anterior pituitary. Proc. Soc. Exp. Biol. Med. 130:406, 1969.

 

Miyachi, Y., R.S. Mecklinburg and M.B. Lipsett: 12_vitro studies of
pituitary median eminence unit. Endocrinology 93:492-496, 1973.

 

Moir, A.T. and D. Eccleston: The effects of precursor loading in the
cerebral metabolism of 5-hydroxyindoles. 1, Neurochem. 15:1093-
1108, 1968.

Moore, R.Y., A. Heller, R.J. wurtman and J. Axelrod: Visual pathway
mediating pineal response to environmental light. Science 155:
220-223, 1967.

Mori, T., A. Suzuki, T. Nishimura and A. Kambegawa: Evidence for andro-
gen participation in induced ovulation in immature rats. Endocri-

Moss, R.L., and S.M. McCann: Action of luteinizing hormone-releasing
factor (LRF) in the initiation of lordosis behavior in the estrone-
primed ovariectomized female rat. Neuroendocrinology 17:309-318.
1975.

165

Moss, R.L. and S.M. McCann: Induction of mating behavior in rats by
luteinizing hormone-releasing factor. Science 181:177-179, 1973.

Moszkowska, A. and M.N. Des Gouttes: L'action des estraits épiphy-
saires sur la réponse du tractus génital de la ratte exposée a la
lumiére continue. Compt. Rend. Soc. Biol. 156:1750-1757, 1962.

 

Mfiller, E.E., A. Arimura, T. Saito and A.V. Schally: Growth hormone
releasing activity in plasma of normal and hypophysectomized
rats. Endocrinology 80:77-81, 1967.

 

Mfiller, E.E., D. Cocalu and A. Villa: Involvement of brain catechol-
amines in the gonadotropin-releasing mechanisms before puberty.
Endocrinology 90:1267-1276, 1972.

 

Murthy, A.S.K. and A.B. Russfield: Endocrine changes in two strains of
mice exposed to constant illumination. Endocrinology 86:914-917,
1970.

 

Naftolin, F., K. Brown-Grant and C.S. Corker: Plasma and pituitary
luteinizing hormone and peripheral plasma oestradiol concentrations
in the normal oestrous cycle of the rat and after experimental mani-
pulation of the cycle. 1, Endocrinol. 53:17-30, 1972.

 

Nallar, R. and S.M. McCann: Luteinizing hormone-releasing activity in
plasma of hypophysectomized rats. Endocrinology 76:272-275, 1965.

Nallar, R.J., J. Antunes-Rodrigues and S.M. McCann: Effect of proges-
terone on the level of plasma luteinizing hormone (LH) in normal
female rats. Endocrinology 79:907-911, 1966.

 

Nauta, W.J.H.: Hippocampal projections and related neural pathways to
the midbrain in the cat. Brain 81:319-340, 1958.

Nauta, W.J.H. and W. Haymaker: Hypothalamic nuclei and fibre connec-
tions. In: The Hypothalamus, edited by W. Haymaker, E. Anderson
and W.J.H. Nauta, pp. 135-209. Springfield: C.G. Thomas, 1969.

 

Negro-Vilar, A., E. Dickerman and J. Meites: FSH releasing factor
activity in plasma of rats after hypophysectomy and continuous
light. Endocrinology 82:939-944, 1968.

 

Negro-Vilar, A., E. Dickerman and J. Meites: Effects of continuous light
on hypothalamic FSH-releasing factor and pituitary FSH levels in
rats. Proc. Soc. Exp. Biol. Med. 127:751-755, 1968.

 

Nequin, L.G. and N.B. Schwartz: Adrenal participation in the timing of
mating and LH release in the cyclic rat. Endocrinology_88:325-
331, 1971.

Nett, T.M., A,M. Akbar and B.D. Niswender: Serum levels of luteinizing
hormone and gonadotrophin-releasing hormone in cycling, castrated
and anestrous ewes. Endocrinology_94:713-718, 1974.

 

166

Nikitovitch—Winer, M.: The influence of the hypothalamus on luteotropin
secretion in the rat. Mem. Soc. Endocrinol. 9:70-72, 1960.

 

 

Nikitovitch-Winer, M.B.: Induction of ovulation in rats by direct
intrapituitary infusion of median eminence extracts. Endocrino-

Piacsek, B.B. and G.M. Hautzinger: Effects of duration, intensity and
spectrum of light exposure on sexual maturation time of female
rats. Biol._Roprod. 10:380-387, 1974.

Piacsek, B.B. and J. Meites: Stimulation by light of gonadotropin re-
lease from transplanted pituitaries of hypophysectomized rats.
Neuroendocrinology 2:129-137, 1967.

 

Piacsek, B.E., T.C. Schneider and V.L. Gay: Sequential study of lu-
teinizing hormone (LH) and "progestin" secretion on the afternoon
of proestrus in the rat. Endocrinology 89:39-45, 1971.

 

Piacsek, B.E. and W.J. Streur: Effect of exposure to continuous light
on estrogen induced precocious sexual maturation in female rats.
Neuroendocrinology 18:86-91, 1975.

 

Pohorecky, L.A., M. Zigmond and H. Karten: Enzymatic conversion of
norepinephrine to epinephrine by the brain. 1, Pharmacol. Exp.
Ther. 165:190-195, 1969.

 

Popa, G.T. and U. Fielding: A portal circulation from the pituitary to
the hypothalamic region. 1, Anat. (London) 65:88-91, 1930.

Porter, J.C.: Hypophysiotropic hormones in portal vessel blood. In:
Hypophysiotrophic Hormones o£_the Hypothalamus: Assay and Chemistry,
edited by J. Meites, Proceedings of the Workshop Conference, Tucson,
Arizona, Baltimore: Williams and Wilkins, p. 282, 1969.

 

 

Porter, J.C., I.A. Kamberi and J.G. Ondo: Role of biogenic amines and
cerebrOSpinal fluid in the neurovascular transmittal of hypophysio-
tropic substances. In: Brain-Endocrine Interaction. Median Emi-
nence: Structure and Function, edited by K,M. Knigge, D.E. Scott
and A. Weindl, Basel: Karger, pp. 245-253, 1972.

 

 

 

Porter, J.C., R.S. Mical and O.M. Cramer: Effect of serotonin and other

indoles on the release of LH, FSH and prolactin. Gynecol. Invest.
2:13-22, 1971.

Porter, J.C., R,S. Mical, P.R. Tippit and J.W. Drane: Effect of selective
surgical interruption of anterior pituitary's blood supply on ACTH
release. Endocrino1ogy_86:590, 1970.

 

Proudht, O.M. and N.B. Schwartz: Reversal of pentobarbital blockade of
ovulation after cardiac puncture. Endocrinology 94:526-531, 1974.

 

167

Psychoyos, A.: Effet de la photopériodicité et de diverses, substances
sur la réponse de l'ovaire a l'hormone luteinisante hypophysaire.
Compp. Rend. Acad. Sci. 263:986-989, 1966.

 

 

Quadri, S.K., G.S. Kledzik and J. Meites: Reinitiation of estrous cycles
in old constant estrous rats by central acting drugs. Neuroendo-
crinology 11:248-255, 1973.

Quay, W.B.: Effect of dietary phenylalanine and tryptophan on pineal and
hypothalamic serotonin levels. Proc. Soc. Exp. Biol. Med. 114:718,
1963.

 

 

Quay, W.B.: Differences in circadian rhythms in 5-hydroxytryptamine
according to brain region. 1m, 1, Physiol. 215:1448-1453, 1968.

Quay, W.B.: The role of the pineal gland in environmental adaptation.
In: Physiology and Pathology 2: Adaptation Mechanisms, edited by
E. Bajusz. New York: Pergamon Press, p. 508-550, 1969.

 

Quay, W.B.: Endocrine effects of the mammalian pineal. 1m, Zoologist
10:237-246, 1970.

Quijada, M., P. Illner, L. Krulich and S.M. McCann: The effect of cate-
cholamines on hormone release from anterior pituitaries and ventral

hypothalami incubated 12_vitro. Neuroendocrinology 13:151—163,
1973/74.

 

Raisman, G. and P. Field: Anatomical considerations relevant to the
interpretation of neuroendocrine experiments. In: Frontiers 12_
Neuroendocrinology, edited by L. Martini and W.D. Ganong, pp. 3-44,
New York: Oxford University Press, 1971.

 

Ramaley, J.A. and D. Bartosik: Rhythms of progesterone and corticoster-
one in the PMS-treated rat: Relationship to ovulation. Neuroendo-
crinology 17:319-329, 1975.

Ramirez, V.D., R.M. Abrams and S.M, McCann: Effect of estradiol implants
in the hypothalamo-hypophsial region of the rat on the secretion of
luteinizing hormone. Endocrinology 75:243-248, 1964.

 

Ramirez, V.D. and C.H. Sawyer: Fluctuations in hypothalamic LH-RF
(luteinizing hormone-releasing factor) during the rat estrous cycle.
Endocrinology 76:282-289, 1965.

 

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

Redding, T.W. and A.V. Schally: Studies on the thyrotropin-releasing
hormone (TRH) activity in peripheral blood. Proc. Soc. Exp. Biol.
Med. 131:420-425, 1969.

 

168

Reiss, M., R.H. Davis, M.B. Sideman, I. Mauer and E.S. Plichta: Action
of pineal extracts on the gonads and their function. 1, Endocrinol.
27:107-118, 1963.

 

Reiter, R.J.: Pineal regulation of hypothalamic pituitary axis: gonado-
tropins. In: Handbook 2: Physiology, Section 2, Part 1, Endocri-
nology 4:519-550, 1974.

Reiter, R.J. and F. Fraschini: Endocrine aspects of the mammalian pineal
gland: A review. Neuroendocrinology 5:219-255, 1969.

 

Reiter, R.J. and S. Sorrentino, Jr.: Reproductive effects of the mammalian
pineal. 12, Zoologist 10:247—258, 1970.

Resko, J.A.: Endocrine control of adrenal progesterone secretion in the
ovariectomized rat. Science 164:70-71, 1968.

Robson, J.M. and M. Botros: The effect of 5-hydroxytryptamine and of
monoamine oxidase inhibitors on sexual maturity. 1, Endocrin. 22:
165-175, 1961.

Ross, G.T., G.M. Cargille, M.B. Lipsett, P.L. Rayford, J.R. Marshall,
C.A. Stot and D. Rodbard: Pituitary and gonadal hormones in women
during spontaneous and induced ovulatory cycles. Recent Progr.
Horm. Egg. 26:1-48, 1970.

 

Ruberstein, L. and C.H. Sawyer: The role of catecholamines in stimulating
the release of pituitary ovulating hormones in the rat. Endocri-
nology 86:990-995, 1970.

Saavedra, J.M., M. Brownstein and J. Axelrod: A specific and sensitive
enzymatic-isotopic microassay for serotonin in tissues. 1,
Pharmacol. Exp. Ther. 186:508-515, 1973.

Saavedra, J.M., M. Palkovits, M.J. Brownstein and J. Axelrod: Serotonin
distribution in the nuclei of the rat hypothalamus and preoptic
region. Brain Res. 77:157, 1974.

Saffran, M. and A.V. Schally: Release of corticotrophin by anterior
pituitary tissue 1p_vitro. Can. 1, Biochem. Physiol. 33:408-415,
1955.

Sawyer, C.H.: First Geoffrey Harris Memorial Lecture. Some recent
developments in brain-pituitary-ovarian physiology. Neuroendocri-
nology 17:97-124, 1975.

Schally, A.V., A. Arimura, C.Y. Bowers, A.J. Kastin, S. Sawano, and T.W.
Redding: Hypothalamic neurohormones regulating anterior pituitary
function. Recent Progr. Horm. Res. 24:497-588, 1968.

Schally, A.V., A. Arimura and A.J. Kastin: Hypothalamic regulatory
hormones. Science 179:341-350.

169

Schally, A.V., E.E. Mfiller and S. Sawano: Effect of porcine growth
hormone-releasing factor on the release and synthesis of growth
hormone 12_vitro. Endocrinology 82:271-276, 1968.

 

Schally, A.V., A. Arimura, Y. Baba, R.M.B. Nair, H. Matsuo and T.W.
Redding: Isolation and properties of the FSH and LH-releasing hor-
mone. Biochem. Biophyg, Res. Comm. 43:393-399, 1971.

 

Schanberg, S.M.: A study of transport of 5-hydroxytryptophan and 5-
hydroxytryptamine (serotonin) into brain. 1, Pharmacol. Exp. Ther.
139:191, 1963.

 

Schapiro, S., J. Marmorston and H. Sobel: Pituitary stimulating substance
in brain blood of hypophysectomized rat following electric shock
'stress'. Proc. Soc. Exp. Biol. Med. 91:382-386, 1956.

 

Scharrer, E. and B. Scharrer: Hormones produced by neurosecretory cells.
Recent Progr. Hormone Res. 10:183-232, 1954.

 

Schnaitman, C., V.G. Erwin and J.W. Greenwalt: The submitochondrial lo-
calization of monoamine oxidase. An enzymatic marker for rat liver
mitochondria. 1, Cell. Biol. 32:719-735, 1967.

 

Schneider, H.P.G. and S.M. McCann: Possible role of dopamine as trans-
mitter to promote discharge of LH-releasing factor. Endocrinology
85:121—132, 1969a.

 

Schneider, H.P.G. and S.M. McCann: Stimulation of release of luteinizing
hormone-releasing factor from hypothalamic tissue by dopamine lo
vitro. 1, Reprod. Fertility 18:178, 1969b.

 

Schneider, H.P.G. and S.M. McCann: Mono- and indolamines and control of
LH secretion. Endocrinology 86:1127-1133, l970a.

 

Schneider, H.P.G. and S.M. McCann: Release of LH-releasing factor into
the peripheral circulation of hypophysectomized rats by dopamine
and its blockage by estradiol. Endocrinology 87:249-253, 1970b.

 

Schneider, H.P.G. and S.M. McCann: Estradiol and the neuroendocrine
control of LH release gp_vitro. Endocrinology 87:330-338, l970c.

 

Schneider, H.P.G. and S.M. McCann: Luteinizing hormone-releasing factor
discharged by dOpamine in rats. 1, Endocrinol. 46:401-402, l970d.

Schreiber, V., A. Eckertova, Z. Franz, J. Koci, M. Rybék and V. Kmentova:
Effect of a fraction of bovine hypothalamic extract on the release
of TSH by rat adenohypophysis gp_vitro. Experientia (Basel) 17:
264-265, 1961.

 

Schwartz, N.B.: A model for the estrous cycle regulation in the rat.
Recent PrOgr. Horm. Res. 25:1-43, 1969.

170

Scott, P.P. and M.A. Lloyd-Jacob: Reduction in the anoestrus period of
laboratory cats by increased illumination. Nature 184:2022, 1959.

Selmanoff, M.K., M.J. Pranik-Holdway and R.J. Weiner: Concentrations of
dopamine and norepinephrine in discreet hypothalamic nuclei during
the estrous cycle of the rat. Endocrinology 99:326-329, 1976.

 

Sheard, M.G. and G.K. Aghajanian: Stimulation of the midbrain raphé:
Effect on serotonin metabolism. 1, Pharmacol. Exp. Ther. 163:425-
430, 1968.

 

Shaskan, E.G. and S.H. Snyder: Kinetics of serotonin accumulation into
slices from rat brain: Relationship to catecholamine uptake. .1.
Pharmacol. Exp. Ther. 175:404, 1970.

 

Shibusawa, K., S. Saito, K. Nishi, T. Yamamoto, C. Abe and T. Kawai:
Effects of thyrotrophin releasing principle (TRF) after section of
the pituitary stalk. Endocr. Jap. 3:151-157, 1956a.

Shibusawa, K., S. Saito, K. Nishi, T. Yamamoto, K. Tomizawa and C. Abe:
The hypothalamic control of the thyrotroph-thyroidal function.
Endocr. Jap. 3:116-124, 1956b.

 

Sinha, D.K. and J. Meites: Effects of thyroidectomy and thyroxine on
hypothalamic concentration of 'thyrotropin releasing factor' and
pituitary content of thyrotropin in rats. Neuroendocrinology
1:4-14, 1965-66.

 

Sinha, D.K. and J. Meites: Stimulation of pituitary thyrotropin synthesis
and release by hypothalamic extract. Endocrinology 78:1002-1006,
1966.

 

Singh, K.B. and G.S. Greenwald: Effects of continuous light on the re-
productive cycle of the female rat: Induction of ovulation and
pituitary gonadotrophins during persistent oestrus. 1, Endocrinol.

38:389-394, 1967.

 

Siperstein, E.R. and M.A. Greer: Observation on the morphology and
histochemistry of the mouse pituitary implanted in the anterior
eye chamber. 1, Nat. Cancer Inst. 17:569-599.

 

 

Smith, E.R. and J.M. Davidson: Luteinizing hormone releasing factor in
rats exposed to constant light: Effects of mating. Neuroendocri-
nology 14:129-138, 1974.

 

Snell, G.D., E. Fekete, K.P. Hummel and L.W. Law: The relation of mating,

ovulation and the estrous smear in the house mouse to time of day.
Anat. Record 76:39-54, 1940.

 

Sokal, R.R. and F.J. Rohlf: Biometry, San Francisco: W.H. Freeman, p.
239, 1969.

171

Spector, S., R. Gordon, A. Sjoerdsma and S. Udenfriend: End-product
inhibition of tyrosine hydroxylase as a possible mechanism for
regulation of norepinephrine synthesis. Mol. Pharmacol. 3:549-555,
1967.

 

Sridaran, R. and C.E. McCormack: Predicting the time of ovulation in
rats by monitoring running activity. Fed. Proc. 36:313, Abstract
N01. 226, 1977.

Stefano, F.J.E. and A.0. Donoso: Norepinephrine levels in the rat hypo-
thalamus during the estrous cycle. Endocrinology 81:1405-1406,
1967.

 

Steger, R.W., J.J. Peluso, J.A. Mitchell and E.S.E. Hafez: Photoperiod
effect of gonadotropin releasing hormone induced ovulation in the
immature rat. Biol. Reprod. 13:597-602, 1975.

 

Stumpf, W.E.: Estrogen-neurons and estrogen-neuron systems in the peri-
ventricular brain. ‘ém, 1, Anat. 129:207-217, 1970.

Stumpf, W.E.: Autoradiographic techniques and the localization of
estrogen, androgen and glucocorticoid in the pituitary and brain.
Amer. Zoologist 11:725-739, 1971.

 

Stumpf, W.E.: Estradiol-concentrating neurons: Topography in the hypo-
thalamus by dry mount autoradiography. Science 162:1001-1003, 1968.

Swerdloff, R.S., B.S. Jacobs and W.D. Odell: Synergistic role of pro-
gestogens in estrogen induction of LH and FSH surge. Endocrinology
90:1529-1536, 1972.

 

Symchovicz, S., W.D. Peckham, R.O. Neri, C.A. Korduba and P.L. Perlman:
The effect gp_vitro of purified hypothalamic extract on the growth
hormone content of the rat pituitary. 1, Endocrinol. 35:379-383,
1966.

Szentagothai, J.: Hypothalamic Control o§_the Anterior Pituitary (3rd
ed.). Budapest: Akadémiai Kiad6, 1968.

 

 

Szentagothai, J., B. Flerko, B. Mess and B. Halasz: Hypothalamic Control
.2£ the Anterior Pituitary. Akadémiai Kiad6, Budapest, 1962.

 

 

Szentagothai, J., B. Flerk6, B. Mess and B. Halasz: Hypothalamic Control

 

o£_the Anterior Pituitary. Budapest: Akadémiai Kiad6, 1968.

 

Tagliomonte, H., P. Tagliomonte, J. Forn, J. Perez-Cruent, G. Krishna
and G.L. Gessa: Stimulation of brain serotonin synthesis by dibu-
tyryl-cyclic AMP in rats. 1, Neurochem. 18:1191-1196, 1971.

Takahashi, M., J.J. Ford, K. Yoshinaga and R.O. Creep: Ovulation in
light estrous rats induced by darkness. Endocrinology Jap. 24:89-
96, 1977.

 

172

Takahashi, M., K. Homma and Y. Suzuki: Resumption of cyclicity in light-
induced persistent estrus by treatment with serotonin or its pre-
cursor. Endocrinol. Jap. 20:271-276, 1973.

 

Takahashi, M. and Y. Suzuki: The dependency of the rat estrous cycle on
the daily alternation of light and dark. Endocrinol. Jap. 16:87-
102, 1969.

 

Tajasen, T. and K.E. Moore: Surgical analysis of the preopticotuberal
pathway controlling ovulating release of gonadotropins in the rat.
Endocrinology 81:1387, 1971.

 

Taleisnik, S., M.B. Velasco and J.J. Astrada: Effect of hypothalamic
deafferentation on the control of luteinizing hormone secretion.
1, Endocrinol. 46:1-7, 1970.

 

Talwalker, P.K., A. Ratner and J. Meites: lp_vitro inhibition of pitui-
tary prolactin synthesis and release by hypothalamic extract.

Terry, W.A. and J. Meites: The effects of continuous light or darkness
on thyroid and reproductive function in ewes. 1, Animal Sci. 10:
1081-1082, 1951.

 

Thomson, A.P.D.: Relation of retinal stimulation to oestrus in the
ferret. ‘1, Physiol. (London) 113:425-433, 1951.

Thorington, L., L. Cunningham and J. Parascandola: The illuminant in
the prevention and phototherapy of hyperbilirubinemia. Ill. Eng.
66:240-250, 1971.

Tilney, F.: The development and constituents of the human hypophysis.
Bull. Neurol. Inst. N.Y. 5:387-436, 1936.

 

 

Tima, L., C.P. Trentini and B. Mess: Effect of serotonin on ovulation
induced by pinealectomy in anovulatory frontal-deafferentated rats.
Neuroendocrinology 12:149-152, 1973.

 

Tonge, S.R. and P.M. Greengrass: The acute effects of oestrogen and pro-
gesterone on the monoamine levels of the brain of ovariectomized
rats. Psychopharmacologia 21:374-381, 1971.

 

Truscott, B.L.: Physiological factors in hypophysial-gonadal interaction.
I. Light and the follicular mechanism of the rat. 1, Exp. 2001.
95:291-305, 1944.

Tsafriri, A. and P.F. Kraicer: The time sequence of ovum maturation in
the rat. 1,_Roprod. Fertilipy 29:387-393, 1972.

 

Turgeon, J.L. and C.A. Barraclough: Regulatory role of estradiol in pi-
tuitary responsiveness of luteinizing hormone releasing hormone on
proestrus in the rat. Endocrinology 101:548-554, 1977.

 

173

Uemura, T.: Studies on the biosynthesis and release of ACTH. Endocr.
J p. 15:130-131, 1968.

Uemura, H. and H. Kobayashi: Effects of dopamine implanted in the
median eminence on the estrous cycle of the rat. Endocrinol. Jap.
18:91, 1971.

 

Ungerstedt, U.: Stereotaxic mapping of the monoamine pathways in the
rat brain. Acta Physiol. Scand. 367:1-48, 1971.

 

Van der Werff ten Bosch, J.J.: Effects of cerebral lesions, blinding
and domestication on gonadal functions of the female ferret. 1,
Endocrinol. 26:113-124, 1963.

 

VanLoon, C.R., and C.L. Kragt: Effect of dopamine on the biological
activity and 12_vitro release of ACTH and FSH. Proc. Soc. Exp.
Biol. Med. 133:1137-1141, 1970.

 

Vaughan, M.K., B. Benson and J.T. Norris: Inhibition of compensatory
ovarian hypertrophy in mice by 5-hydroxytryptamine and melatonin.
1, Endocrinol. 47:397-398, 1970.

 

Vaughan, M.K., G.M. Vaughn and W.K. O'Steen: Oestrous cycle changes
after neonatal injections of 5-hydroxytryptamine in normal and
androgenized female rats. ‘1, Endocrinol. 45:141-142, 1969.

 

Vernikos-Danellis, J.: Effect of rat median eminence extracts on pitui-
tary ACTH content in normal and adrenalectomized rats. Endocrino-
logy 76:240-245, 1965.

Waddill, D.G., C.H. Chaney and R.H. Dutt: Ovulation rate in gilts after
short time exposure to continuous light. 1, Reprod. Fertiligy 15:
123-125, 1968.

 

Weick, R.F., E.R. Smith, R. Dominguez, A.P.S. Dhariwal and J.M. Davidson:
Mechanism of stimulatory feedback effect of estradiol benzoate on
the pituitary. Endocrinology 88:293-301, 1971.

 

Weiner, N. and M. Rabadjija: The regulation of norepinephrine synthesis:
Effect of puromycin on the accelerated synthesis of norepinephrine
associated with nerve stimulation. {1, Pharmacol. Exp. Ther. 164:
103-114, 1968.

 

Weiner, R.I.: Hypothalamic monoamine levels and gonadotropin secretion
following deafferentation of the medial basal hypothalamus. Prog.
Brain Res. 39:165-170, 1973.

Weiner, R.I., R.A. Gorski and C.H. Sawyer: Hypothalamic catecholamines
and pituitary gonadotropic function. In: Brain-Endocrine Inter-
action, Median Eminence: Structure and Function, edited by K.M.
Knigge, D.E. Scott and A. Weindl, Basel: Karger, pp. 236-244, 1972.

 

 

 

174

Weiner, R.I., J.E. Shryre, R.A. Gorski and C.H. Sawyer: Changes in the
catecholamine content of the rat hypothalamus following deafferen-
tation. EndocrinOIOgy 90:867-873, 1972.

 

Weissback, H., B.G. Redfield and J. Axelrod: The enzymatic acetylation
of serotonin and other naturally occurring amines. Biochem.
Biophys. Acta 54:190-192, 1961.

 

Wheaton, J.E., L. Krulich, and S.M. McCann: Localization of luteinizing
hormone-releasing hormone in the preoptic area and hypothalamus
of the rat using radioimmunoassay. Endocrinology_97:30-38, 1975.

 

Wheaton, J.E., S.K. Martin, L.S. Swanson and F. Stormshak: Changes in
hypothalamic biogenic amines and serum LH in the ewe during the
estrous cycle. 1, Animal Sci. 35:801-804, 1972.

 

Wilber, J.F. and J.C. Porter: Thyrotropin and growth hormone releasing
activity in hypophysial portal blood. Endocrinology 87:807-811,
1970.

 

Wilson, C., C. Endersby and P.G. MacDonald: Potentiating effect of sero-
tonin on pregnant mare serum gonadotrophin induced ovulation in
the underweight immature rat. 1, Endocrinol. 6l:xxv (Abstract),
1973.

 

Wilson, C. and P.G. MacDonald: Inhibitory effect of serotonin on
ovulation in adult rats. 1, Endocrinol. 60:253-260, 1974.

 

Wislocki, G.B. and L.S. King: The permeability of the hypophysis and
hypothalamus to vital dyes, with a study of the hypophyseal vascular
supply. ‘19, 1, Anat. 58:421-472, 1936.

Wurtman, R.J.: Effects of light and visual stimuli on endocrine func-
tion. In: Neuroendocrinology, edited by L. Martini and W.F.
Ganong. New York: Academic Press, Vol. II, p. 19-59, 1967.

 

Wurtman, R.J. and J. Axelrod: Central control of the pineal gland:
Visual pathways. Arch. Neurol. 18:208-218, 1968.

 

Wurtman, R.J., J. Axelrod, E.W. Chu and J.E. Fischer: Mediation of some
effects of illumination on the estrous cycle by the sympathetic
nervous system. Endocrinology 75:266-272, 1964.

Wurtman, R.J., J. Axelrod and D.E. Kelly: The Pineal. New York:
Academic Press, 1968.

 

Wurtman, R.J. and J. Weisel: Environmental lighting and neuroendocrine
function: Relationship between spectrum of light source and
gonadal growth. Endocrinology 85:1218-1221, 1969.

 

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

 

175

Wuttke, W. and J. Meites: Effects of electrochemical stimulation of
hypothalamus in serum prolactin and LH in rats. Pflogers Arch.
337:71, 1972.

 

Yamamoto, M., N.D. Diebel and E.M. Bogdanove: Analysis of initial and
delayed effects of orchidectomy and ovariectomy on pituitary and

serum LH levels in adult and immature rats. Endocrinology 86:
1102-1111, 1970.

 

Ying, S.Y. and R.0. Greep: Inhibition of ovulation by melatonin in the
cyclic rat. Endocrinology 92:333-335, 1973.

 

Zeilmaker, C.H.: The biphasic effect of progesterone on ovulation in
the rat. Acta Endocrinol. (Kbh.) 51:461-468, 1966.

 

Zolovick, A.J.: Role of central sympathetic neurones in the release of
gondadotropin after hemiovariectomy. 1, Endocrinol. 52:201-202,
1971.

 

Zolovick, A.J.: Effects of lesions and electrical stimulation of the
amygdala on hypothalamus-hypophseal regulation. In: 1hg_
Neurobiology_o£_£hg_Amygdala, edited by E.E. Eleftheriou, New York:
Plenum Press, pp. 643-683, 1972.

 

Zolovick, A.J., and A.P. Labhsetwar: Letter: Evidence for the theory
of dual hypothalamic control of ovulation. Nature 245:158-159,
1973.

Zscharek, L.L. and R.J. Wurtman: Brain H-catechol synthesis and the
vaginal estrous cycle. Neuroendocrinology 11:144-149, 1973.