AN ABSTRACT OF THE THESIS OF
Salaheldin Eltigani Abdelgadir for the degree of Doctor of Philosophy
in Animal Science
presented on
July 31, 1987.
Title: OXYTOCIN SYNTHESIS AND RELEASE BY THE BOVINE CORPUS LUTEUM
Abstract approved:
Redacted for privacy
Dr. Fredrick Stormshak
Redacted for privacy
Abstract approved:
D1k-.
Four experiments were conducted to
tration
of
bovine
concen
oxytocin
determine
luteal tissue at different stages of the estrous
cycle and to study the effects
(PGE2),
(dfield
James E.
luteinizing
hormone
of
(LH),
(PGF2a),
F2a
prostaglandins
colchicine
cycloheximide,
release
vitro.
E2
and
In
cytochalasin B
on
experiment
luteal oxytocin concentrations (ng/g) in beef heifers
1,
oxytocin
synthesis
and
increased from 414 + 84 on day 4 to 2019 + 330
declined
to
589
on
in
day
8
and
then
+ 101 on day 12 and 81 + 5 on day 16 of the cycle.
Prostaglandin F2a induced a significant in vitro
oxytocin on day 8 but not on days 12 or 16,
release
of
luteal
while PGE2 and LH had no
studied.
effect on oxytocin release at any stage of the cycle
Total
oxytocin concentration (incubation medium + tissue) increased twofold
In experiment 2, six beef heifers were used
after 2 h of incubation.
investigate
to
vitro
in
effects
20 and 40 ng PGF2a /ml of
of 10,
linear
doseresponse
release
relationship
dosedependent.
was
In
significant
A
tissue.
medium on oxytocin release from day 8 luteal
was observed indicating oxytocin
experiment
effects
the
3,
of
cycloheximide on oxytocin synthesis as well as PGF2ainduced oxytocin
Although,
from day 8 bovine luteal tissue was investigated.
release
cycloheximide inhibited incorporation of labeled leucine into protein
by more than 90%,
prostaglandin
tissue.
F2ainduced
In
experiment
processing
affect
release
incorporation
No
oxytocin.
it did not
of
or
this nonapeptide from luteal
of
labeled
the
4,
prohormone
of
leucine
cytochalasin
of
effects
detected
was
in
and
B
colchicine on oxytocin synthesis and release from day 8 bovine luteal
tissue were
inhibited
investigated.
oxytocin
synthesis
nor
colchicine
Neither
B
caused
a
PGF2a
while
release
or
cytochalasin
significant release of oxytocin that was not inhibited by colchicine.
These studies indicate that maximal oxytocin concentrations in bovine
luteal tissue occur during the
Luteal
PGE
2
oxytocin
early
phase
luteal
translational
due
to
short
processing
occurred in the absence
of
cycle.
the
in vitro can be induced by PGF2a whereas
secretion
and LH have no effect on oxytocin secretion.
concentration
of
term
of
an
incubation
represents
prohormone
oxytocin
incorporation
Increased oxytocin
of
during inhibition of de novo protein synthesis.
labeled
a
post
because
leucine
it
and
Oxytocin Synthesis and Release by
the Bovine Corpus Luteum
by
Salaheldin Eltigani Abdelgadir
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed July 31, 1987
Commencement June, 1988
APPROVED:
Redacted for privacy
Professor of Animal Science in charge of major
Redacted for privacy
Profes o
of Animal
lence
in charge of major
Redacted for privacy
c_X
Head of Department of Animal Science
Dean of Graduate School
Date thesis is presented
July 31, 1987
To my wife Afaf, for her immense patience,
magnificent devotion, encouragement and love,
and to my son Mohamed and my daughter Reim,
for making life so wonderful and enjoyable,
I dedicate this thesis
ACKNOWLEDGEMENTS
Foremost among the people
who
contributed
of this program are Dr.
completion
successful
the
to
Fred Stormshak and Dr.
James E.
Oldfield, my major professors.
My deepest gratitude and sincere appreciation
Stormshak
allowing
for
laboratory under
meticulous
undertake
to
advice.
his
expertise were invaluable.
work,
me
His
trust,
go
to
research
this
fairness,
Fred
Dr.
in
his
guidance
and
He bestowed on me an appreciation of hard
experimentation,
and
the desire to think and ask
questions about science and life in general.
Oldfield
James
Dr.
His confidence in my cabability as a scientist meant a
endless help.
lot to me.
to
sound advice and
his tremendous encouragement,
for
go
also
My deepest gratitude and sincere appreciation
He provided me with assistantship when I needed it
most.
He was understanding and he was there for me whenever I needed him.
For
this
all
shall
I
be
indebted
Oldfield and Dr.
to Dr.
Stormshak forever.
Many thanks
Physiology,
as
Dr.
Dieter
Schams
Technical University of Munich,
the
of
well
technical advice.
as
Institute
of
FreisingWeihenstephan,
for the generous donation of the rabbit
West Germany,
serum,
to
go
antioxytocin
This research would not have
been possible without his help.
Gratitude
Moore,
members
Dr.
is
also extended to Dr.
Steve Davis and Dr.
Wilbert Gamble,
Wilson Schmisseur
for
Dr.
Frank
serving
as
of my graduate committee and for their valuable constructive
criticisms and advice.
Claire Wathes of the Department of Anatomy,
I wish to thank Dr.
University
Bristol,
of
encouragement.
for
England,
and
Lloyd Swanson who helped
are also due to Dr.
Thanks
advice
technical
her
with the development and validation of oxytocin radioimmunoassay
and
Dr.
Ken Rowe,
for assistance with statistical analyses of the data.
Dr.
Dale Weber
,
Department
Marvin Martin and Mr.
Mr.
greatly
are
Bob
Dickson
of
this
for arranging for the supply,
acknowledged
care and slaughter of the experimental animals.
Appreciation
Experiment
is
Station
assistantship.
Many
also extended to the Oregon State Agricultural
for
provision
thanks
and
the
research
are extended to the Sudanese Government
Mahgoub Elbadawi, the Cultural Counsellor of the Sudanese
and to Mr.
Embassy in Washington DC for their
Omer
funding
of
Idris
and
Dr.
Abdelgadir
Research Administration in
immense
of
Wahbi
Khartoum
help
are
and
support.
Dr.
the Sudanese Veterinary
greatly
acknowledged
for
their unlimited support and encouragement.
My colleagues and friends,
Rose, Dr.
Dr.
Mary ZelinskiWooten, Dr.
Jack
Tony Archibong, John Jaeger, Carrie Cosola, Ov Slayden and
Teri Martin deserve my gratitude for their encouragement and
endless
support as well as their help during various stages of my research.
I
will miss them all.
Special thanks and love to my father and
Thoraia,
for
mother,
Eltigani
their great love and support throughout my life.
and
They
never ceased to encourage me to strive towards excellence.
Finally to the most glorious of all, to the holy god who gave me
the wisdom and will to go through this
fate.
I
surrender
myself
and
my
TABLE OF CONTENTS
Page
LITERATURE REVIEW
1
Hormonal Control of Corpus Luteum Function During the
1
Estrous Cycle and Pregnancy
General Features of the Estrous Cycles of the Ewe and Cow
2
Hormonal Control of Folliculogenesis
3
Hormonal Control of the Estrous Cycle in the Ewe and Cow
6
Mechanism of Ovulation
9
Formation of the Corpus Luteum
11
Luteotropic Effect of LH
12
Mechanism of Action of LH
13
Mechanism of Luteolysis
13
Endocrine Regulation of PGF a Secretion
16
2
Embryonic Luteotropins
17
Paracrine Regulation of Luteal Function
21
Cell Types of the Corpus Luteum
21
Synthesis and Secretion of Peptide Hormones by the
26
Corpus Luteum
Oxytocin in The Corpus Luteum
26
Comparison of Luteal and Hypothalamic Oxytocin
27
Variations in Luteal Oxytocin Levels
28
Control of Luteal Oxytocin Secretion
31
Actions of Ovarian Oxytocin
31
GnRHLike Ovarian Hormone
33
Relaxin in the Corpus luteum
38
STATEMENT OF THE PROBLEM
42
EXPERIMENTS 1 AND 2: PROSTAGLANDIN Fla INDUCED
43
RELEASE OF OXYTOCIN FROM BOVINE CORPORA
LUTEA IN VITRO
INTRODUCTION
43
TABLE OF CONTENTS (Cont.)
Page
MATERIALS AND METHODS
45
Experiment 1
45
Experiment 2
46
Oxytocin Extraction
47
Oxytocin Radioimmunoassay
48
Statistical Analyses
49
49
RESULTS
Experiment 1
49
Experiment 2
51
55
DISCUSSION
EXPERIMENTS 3 AND 4: CYCLOHEXIMIDE, COLCHICINE
59
AND CYTOCHALASIN B DO NOT AFFECT BOVINE LUTEAL
OXYTOCIN SYNTHESIS AND RELEASE IN VITRO
INTRODUCTION
59
MATERIALS AND METHODS
60
60
Experiment 3
Experiment 4
61
14
Total Incorporation of [
C]leucine into protein
62
Oxytocin Extraction and Radioimmunoassay
63
Statistical Analyses
63
RESULTS AND DISCUSSION
63
GENERAL DISCUSSION
70
BIBLIOGRAPHY
73
LIST OF FIGURES
Figure
Page
EXPERIMENTS 1 AND 2: PROSTAGLANDIN Fla INDUCED
RELEASE OF OXYTOCIN FROM BOVINE CORPORA LUTEA
IN VITRO
1
Oxytocin released (mean + SE) into medium after
2
PGF2a or
h incubation of luteal tissue with LH,
PGE
on days 8,
52
12 and 16 of the estrous cycle.
2
2
Concentrations of oxytocin (mean +
tissue slices
and
in
tissue
incubation with PGF a on days 8,
2
estrous cycle.
SE)
in
luteal
+ medium after 2 h
12 and 16 of the
53
LIST OF TABLES
TABLE
Page
EXPERIMENTS 1 AND 2: PROSTAGLANDIN Floc INDUCED
RELEASE OF OXYTOCIN FROM BOVINE CORPORA LUTEA
IN VITRO
Oxytocin concentrations (mean
1
+
SE)
in bovine
50
luteal tissue at different stages of the estrous cycle
Oxytocin synthesis and(or) release by luteal tissue
2
54
in response to various levels of PGF a in vitro
2
EXPERIMENTS 3 AND 4: CYCLOHEXIMIDE, COLCHICINE AND
CYTOCHALASIN
B DO NOT AFFECT BOVINE LUTEAL OXYTOCIN
SYNTHESIS AND RELEASE IN VITRO
14
3
Incorporation of [
C]leucine (mean + SE) in bovine
64
luteal tissue in vitro
4
Oxytocin synthesis and(or) release by luteal tissue in
66
response to cycloheximide and PGF a in vitro
2
5
Oxytocin synthesis and(or) release by luteal tissue in
response to colchicine, cytochalasin B and PGF a
2
in vitro.
67
Oxytocin Synthesis and Release by
the Bovine Corpus Luteum
LITERATURE REVIEW
Hormonal Control of Corpus Luteum Function During the Estrous Cycle
and Pregnancy
Understanding the basis for estrous cycles and
of
pregnancy
domestic
in
animals
maintenance
the
requires an appreciation of the
factors that regulate corpus luteum function.
The function of
estrous
corpus
the
luteum
during
ruminants
in
cycle and pregnancy is regulated by a complex interaction of
hormones from several sources, including the pituitary gland,
and
the
mechanisms
function
luteal
on
(luteotropic)
or through indirect
These hormones may act directly,
placenta.
or
and secretion,
and
are
uterus
either
stimulatory
(luteolytic) to progesterone synthesis
inhibitory
which are universally utilized
as
measures
of
the
state of luteal function.
In
addition,
factors synthesized and secreted within the ovary
may influence corpus luteum function.
mechanism
evident.
The existence of
a
paracrine
for regulation of luteal function is becoming increasingly
The corpus luteum
of
domestic
animals
and
primates
is
composed of at least two cell types that differ in their function and
response
to
hormonal
luteal function.
stimuli
and
which
may interact to regulate
2
In this section,
estrous
cycles
as
hormonal regulation of the
well
as
corpus
luteum
bovine
ovine
and
during early
function
pregnancy will be discussed. Special consideration will be devoted to
the review of follicular growth as well as effects and mechanisms
action
luteinizing
of
hormone
(LH),
prostaglandin
of
(PGF2a),
F2a
embryonic luteotropins and paracrine factors on luteal function.
General Features of the Estrous Cycles of the Ewe and Cow
The estrous cycle is shorter in ewes (17 days) than in cows (2122 days).
Duration of standing estrus is 24-36 h in the ewe and
19
the cow.
in
h
18-
Ewes normally ovulate near the end of estrus but
time of ovulation varies from as long as 11 h before to 7 h after the
end of estrus.
Cattle ovulate on the average'at 28-32
begining of estrus,
after
h
the
which normally corresponds to 12 h after the end
of standing estrus.
Estrous cycles in the cow and ewe are characterized
luteal
phase.
This
is
the
luteum resides in the ovary.
period
from
corpus
period
during
by
long
a
which an active corpus
In contrast, the follicular phase,
the
luteum regression to the following ovulation is
apparently short (2 days in ewes and 4 to 5 days in
cows).
However,
the presence of antral follicles throughout the luteal phase suggests
that
the real duration of the follicular phase is longer than 2 to 5
days, if one considers that the follicular phase refers to the period
from antral follicle formation to ovulation.
phase
in
these
species
may
Therefore,
the
luteal
partially overlap the true follicular
3
obscuring the relationship between the hormones that regulate
phase,
luteal function and follicular growth (Hafez et al., 1980).
Hormonal Control of Folliculogenesis
Because follicular growth and recruitment of ovulatory follicles
are
it is deemed appropriate
an integral part of the estrous cycle,
Most of these
to mention the factors that regulate folliculogenesis.
factors are also involved in the regulation of the estrous cycle.
Primordial follicles,
squamous
follicular epithelium,
fetal development.
stage
of
the
first mitotic prophase.
However,
process
during
in
dictyate
This resting stage is called
Primary follicles
adulthood
enter
response
pool
a
of
growing
to an undefined stimulus.
a vast majority of these growing follicles degenerate by
known
as
atresia.
a
are established in the ovary during
After birth oocytes are arrested in the
the dictyate nucleus.
follicles
with their single oocyte surrounded by
a
Of the approximately 150,000 primordial
follicles present at birth in heifers (Erickson, 1966), less than 100
will mature and ovulate during the life time
of
an
average
animal
(Hansel and Convey, 1983).
Primordial
follicles
enter a growing phase when the follicular
cells proliferate and form several layers of granulosa cells.
follicle grows it is displaced toward the center of
theca layer differentiates into two layers;
the oocyte aquires a distinct zona pellucida.
the
As the
ovary,
the
interna and externa, and
Growing follicles form
fluid filled antra by the coalesence of small fluid
filled
cavities
4
between follicular cells (Hansel and Convey, 1983).
Factors
controlling
ovulatory follicle(s)
hormone
are
follicle
and
growth,
not
atresia
selection
(FSH)
hormone
released
are
concomitantly at or near the onset of estrus in cows (Akbar
1974) and ewes (Pant et al.,
preovulatory
gonadotropin
et
the
there is a
before ovulation,
but
al.,
after
1977) and approximately 24 h
surge,
the
of
Luteinizing
understood.
completely
stimulating
and
second increase in serum concentrations of FSH in ewes (Pant
et
al.,
1977) and cows (Dobson, 1978; Ireland and Roche, 1982).
This increase
may
Cahill et al.
play a role in recruitment of preantral follicles.
(1981) found a high correlation between the magnitude of this peak
in
FSH concentration and number of antral follicles present in the
serum
ovary 17 days later.
granulosa
Follicle
also
effect.
the
granulosa
cells
stimulated
In addition,
mitosis and follicular fluid formation.
cell
17f3estradiol produced by
mitotic
hormone
stimulating
enhanced
FSH
this
stimulating hormone also induces granulosa
Follicle
cell sensitivity to LH by increasing the number of LH receptors (Hafez
et al., 1980).
Intraovarian factors may also control the growth
of
follicles and the selection of those destined to ovulate.
the
dictyate
nucleus
ovulatory surge.
oocyte was removed
gonadotropinfree
never
primordial
Meiosis of
resumes normally before a gonadotropin
However, in all mammalian species studied, when the
from
the
medium,
metaphase I or metaphase II,
it
antral
follicle
spontaneously
and
resumed
cultured
in
a
meiosis up to
the stage normally attained at the time
5
of ovulation (Thibault,
or
theca
cells
in
a
state
medium
The
follicular fluid
contained
that
oocyte
the
nucleus
in
a
from the inhibitory effect of the granulosa
resulted
Tsafriri and Channing,
cells on the oocyte (Foote and Thibault, 1969;
1975).
granulosa
Culture of the oocyte with
showed that the maintenance of
extracts,
dictyate
or
1977).
of the LHFSH ovulatory surge is to cause loosening
role
of granulosa cell junctions and to suppress production of
a
meiotic
inhibiting factor by granulosa cells (Hafez et al., 1980).
Removal
of the corpus luteum of cows (Hammond and Bhattacharya,
1971) resulted in ovulation in
1944) and ewes (Smeton and Robertson,
48 to 72 h.
of
corpus
Roche,
develop
1982)
and
become
subsequently
and
it
ovulate.
recruited
Removal of the corpus
inhibitor,
eliminate a local
increase gonadotropin stimulation leading to final growth
may
maturation
coincided
newly
while
atretic,
luteum or its regression may therefore,
or
1971) or regression
luteum removal (Smeton and Robertson,
(Ireland and
follicles
large follicles present in ovaries at the time
However,
with
of
the
luteal
follicles,
preovulatory
regression
and
a
whose
development
preovulatory gonadotropin
surge.
Intraovarian factors may interact
hypothalamic
ovulation.
and
other
factors
to
within
regulate
themselves
and
with
folliculogenesis and
Synthesis of hypothalamic hormones and their receptors in
ovarian tissue as well as the presence
ovary that bind gonadotropin receptors,
factors that inhibit FSH secretion
give
of
unique
peptides
in
the
and the existence of uterine
credence
to
this
concept
6
(Hansel and Convey, 1983).
Hormonal Control of the Estrous Cycle in the Ewe and Cow
The estrous cycle is controlled by the interaction of
estrogen
progesterone.
and
domestic animals;
effects
vary
however,
These
species.
LH,
to
most
common
are
patterns
their secretory
different
among
hormones
FSH,
relative
and
differences lead to
These
variations in the length of follicular and_luteal phases of the cycle
as well as differences in duration of estrus.
Patterns of hormonal changes during the estrous cycle in the ewe
and
cow
are
similar.
The
phase
follicular
characterized by rapidly decreasing levels
luteal
regression,
is
due
to
progesterone
which may be important in inducing follicular
the proestrous rise in estradiol (Hansel and Convey,
and
Estradiol increased in ovarian venous (Bjersing et al., 1972;
1983).
Baird and Scaramuzzi,
1971;
cycle
a peak of estradiol and a slight but significant
increase in LH levels,
maturation
of
the
of
1976) and in systemic blood (McCracken et al.,
Hansel et al., 1973) during the preovulatory period reaching a
peak during estrus.
progesterone
positive
resulting
the
and
feedback
in
the
feedback
Removal of the negative
increase
effect
on
ovulatory
in
the
surge
of
estradiol concentration exert a
hypothalamushypophyseal
of
LH
and
approximately 12 h after the onset of estrus in the
and Dunn, 1980;
influence
Hansel and Convey, 1983).
FSH
ewe
that
axis
occurs
(Kaltenbach
The increase in estradiol
that occurs during the preovulatory period is
clearly
the
stimulus
7
that
triggers the gonadotropin surge.
Exogenous estradiol induced a
preovulatorylike surge of LH in ewes (Howland et al., 1971) and cows
(Beck and Convey,
inhibition
of
1977).
In
chemical
addition,
immunological
or
estradiol at proestrus abolished the LH surge in ewes
However, a
(Fairclough et al., 1976) and cows (Martin et al., 1978).
decrease in progesterone is requisite for an estradiol effect on
gonadotropin
Estradiol
surge.
exert
not
did
the
a positive feedback
effect in females bearing a maximally functional corpus luteum
(Bolt
et al., 1971; Short et al., 1973), and exogenous progesterone blocked
the
estradiolinduced gonadotropin surge in ewes (Scaramuzzi et al.,
1971) and heifers (Kesner et al., 1981).
Nevertheless, the mechanism
by which estradiol induces a gonadotropin
understood.
Exogenous
estradiol
increased
pituitary gland to release LH and FSH
releasing
hormone
Kesner et al.,
(GnRH)
in
surge
ewes
is
capacity
the
response
in
completely
not
to
and cows (Reeves et al.,
This capacity was greatest during estrus
1981).
gland
1971;
and
Estradiol
has the ability to prime the response of the anterior pituitary
to
quantity
subsequent
exposures
responsible,
in
GnRH,
Hansel and Convey, 1983).
thereby
increasing
the
increased
vitro,
the
an
during
ability
effect
This priming effect was
for the marked increase in pituitary
at least in part,
sensitivity that occurred
markedly
to
of LH and FSH released by a standard dose of GnRH (Crighton
and Foster, 1977;
cells
the
gonadotropin
least during the luteal phase of the cycle (Convey, 1973).
also
of
proestrus
the
of
that
period.
Estradiol
GnRH to prime bovine pituitary
is
inhibited
by
progesterone
8
(Padmanabhan
et
1982).
al.,
pulsatile releases of GnRH,
increased
preovulatory
surge
of
of
Both
LH.
pulses
thereby
increased
GnRH
LH
to
progressively
under estrogen dominance,
magnitude
the
exposures
consecutive
Moreover,
creating
the
secretion
and
increased pituitary responsiveness are necessary for the preovulatory
LH
and
surges
FSH
(Kesner
Convey,
and
preovulatory LH and FSH surges results
Termination
1982).
refractoriness
from
of
the
of
pituitary gland to GnRH (Chakraborty et al., 1974; Kesner and Convey,
1982)
and
to depletion of gonadotropin content (Convey et al.,
not
1981).
resulted
Increased LH concentrations during the presurge period
from
increased
frequency
and
decreased
amplitude
secretion of this gonadotropin in ewes (Baird,
et al.,
1978) and cows
(Rahe
Each pulse of LH
was
followed
by
an
concentration of estradiol in ovarian venous blood of ewes
(Baird et al.,
1976) and exogenous LH increased estradiol
from autotransplanted ovaries (McCracken et al., 1971).
follicles
pulsatile
These pulses of LH may stimulate estradiol secretion
1980).
from preovulatory follicles.
increased
in
have
been
assessed
as
estrogen inactive (atretic) based
secretion
Preovulatory
estrogen active (nonatretic) and
upon
histological
assessment
of
granulosa cells and steroid hormone content of follicular fluid (Moor
et
al.,
Carson et al.,
1978;
both estrogen active
ovaries,
but
by
follicles remain.
the
and
1981).
inactive
time
of
Following luteal regression,
follicles
the LH surge,
are
present
on
the
only estrogen active
Ovulatory follicle(s) grow in size and the
number
of LH receptors in theca and granulosa cells increases.
these
Consequently
follicles become more responsive to LH and aquire an increased
ability to secrete estradiol.
During
heifers,
active follicle develops and all other
single
a
estrogen
the
estrogen active follicles regress (Ireland
follicle
postovulatory
and
period
This
1983).
Roche,
in
probably the source of increased estradiol secretion at
is
this time of the estrous cycle (Glencross et
al.,
1973;
Hansel
et
al., 1973).
Mechanism of Ovulation
Preovulatory follicles undergo three major
ovulatory
process.
cohesiveness among
maturation
of
These
the
the
oocyte
disruption
include:
granulosa
changes
layer,
of
cytoplasmic
during
the
cumulus
cell
nuclear
and
and thinning and rupture of the external
follicular wall.
The
freeing of the oocyte inside the follicle is the only known
response directly
dissociation
of
(Thibault et al.,
to
attributable
to
gonadotropic
action.
In
vitro
cumulus cells is exclusively obtained by FSH and LH
1975).
The role of the LHFSH ovulatory surge
cause loosening of granulosa cell junctions,
is
suppress production
of oocyte meioticinhibiting factor by granulosa cells and allow
the
oocyte to resume the meiotic division as discussed previously.
The preovulatory gonadotropin surge also induces ovulation by
cascade
of
biochemical
changes.
It
has
been
immediate and temporary rise in steroid levels due
to cause an
shown
to
a
an
increased
10
secretion
progesterone.
of
secretion was
also
Later PGF2a and prostaglandin E2 (PGE2)
augmented.
Inhibition
of
either
progesterone
(Lipner and Greep, 1971) or prostaglandin (Armstrong, 1975) synthesis
prevented ovulation.
ratio
the
in
Enhancement of steroid secretion and the change
of
estradiol
to
that
progesterone
follow
the
gonadotropin surge were easily detectable in follicular fluid (Gerard
et al.,
These changes
1979).
were
barely
detectable
ovarian
in
venous blood and undetectable in systemic blood (Eiler and Nalbandov,
1977).
It
has
been
postulated that the role of progesterone is to
stimulate the activity of collagenase and other
(Rondell,
1970),
enzymes
proteolytic
degrade connective tissue in the follicular
which
wall.
Prostaglandins
play
basic
a
role in follicular rupture,
their action is exerted at the level of the albuginea and
epithelium.
They
ovulation
induce
increasing
by
and
follicular
the activity of
proteolytic enzymes that cause rupture of the follicle and release of
the oocyte
(Espey,
1978).
Inhibition
of
synthesis
prostaglandin
prevented ovulation but not luteinization (Yang et al., 1973;
1974;
al.,
Armstrong and Zamecnik,
1975).
As a result the
Lau et
oocyte
remained inside the luteinized follicle (Osman and Dullaart, 1976).
Prostaglandin Fla, stimulated synthesis,
of
a
collagenaselike
LeMaire and Marsh,
lysosomes
of
ovulatory
1975).
release and
activation
enzyme (Marsh and LeMaire,
1973;
It also contributed to the rupture of the
epithelial cells at the follicular apex and stimulated
the production of
plasminogen
activator,
thus
increasing
plasmin
11
activity
that converted procollagenase to collagenase (Espey,
and is generally involved in cell migration and mixing of
1980)
theca
and
granulosa cells during corpus luteum formation.
Prostaglandin
E2 has also been shown to stimulate production of
plasminogen activator (Strickland and Beers, 1976) and the remodeling
of follicular layers,
terminating in corpus luteum formation
(Hafez
et al., 1980).
Formation of the Corpus Luteum
thickens
Following ovulation the wall of the follicle gradually
due
to hypertrophy and hyperplasia of the theca and granulosa cells.
Rapidly proliferating cells fill the remaining cavity
secrete
progesterone.
The
resulting
corpus
luteum
begin
to
continues
to
and
increase in size and weight and attains full growth and function
days
after
ovulation
in the ewe (Duncan et al.,
7-9
1960) and 12 days
postovulation in the cow (Erb et al., 1971).
The
size
ability
to
of
the
secrete
hydroxyprogesterone
Armstrong,
1973)
luteum is highly correlated with its
corpus
progesterone.
content
of
bovine
Progesterone
corpus
luteum
(Hafs
1968) and progesterone in systemic blood (Hansel et
increase
to
maximal
levels
at
approximately
Progesterone prepares the endometrium for implantation and
pregnancy.
200
and
day
and
al.,
10.
maintains
12
Luteotropic Effect of LH
first
The
that LH possessed luteotropic properties
indication
was provided by Mason et al.
effect
on
(1962) who demonstrated its stimulatory
vitro progesterone synthesis by bovine luteal slices.
in
Since then much evidence has accumulated indicating that
LH
the
is
luteotropic hormone in both the cow and ewe (see Hansel et al., 1973;
Rothchild,
Niswender et al.,
1981;
Exogenous LH injections
1985).
1965b) and
prolonged the life span of bovine (Donaldson and Hansel.,
cycle,
1971) corpora lutea during the estrous
ovine (Karsch et al.,
increased progesterone concentration in the plasma of hysterectomized
ewes
cows
and
prevented the
during
produced
et al.,
1969;
luteolytic
effects
of
bovine
the
addition,
(Brunner
estrous
simultaneous injections of a potent
mares
in
caused
and
inhibited
administered
et
early
1965).
al.,
antibovine
LH
In
serum
a significant reduction in corpus luteum
weight and progesterone content in
1969)
oxytocin
(Donaldson
cycle
1971) and
Carlson et al.,
stimulatory
intact
heifers
of
effects
LH
(Snook
on
et
al.,
progesterone
synthesis (Hansel, 1971).
Furthermore, the secretion of progesterone depends on continuous
luteotropic support from the pituitary gland.
and
secretion
ceased
and
the corpus luteum regressed within a few
days following hypophysectomy in the
the
Progesterone synthesis
ewe.
However,
maintenance
corpus luteum in hypophysectomized pregnant and nonpregnant ewes
occurred if crude pituitary extracts containing LH and
were
of
constantly
infused (Kaltenbach et al.,
1968a,b).
FSH
activity
In addition
13
but
LH,
secretion
not
in
prolactin
or
markedly
FSH
increased
progesterone
autotransplanted ovaries in the ewe (McCracken et al.,
1971).
Mechanism of Action of LH
At the molecular level LH exerts
cascade
action
its
by
initiating
a
of biochemical reactions that lead to increased progesterone
synthesis and the expression of a functional
corpus
(for
luteum
a
recent review see Stormshak et al., 1987).
Luteinizing hormone binds to its receptor in the plasma membrane
of the luteal cell.
convertes
This binding activates adenylate
adenosine
triphosphate
(ATP)
cyclase
which
cyclic adenosine 3'-5'
to
monophosphate (cAMP) which in turn activates a cAMPdependent protein
kinase.
Protein
kinase
turn
in
phosphorylation
causes
of
steroidogenic
enzymes and other proteins necessary for synthesis and
secretion
progesterone.
of
internalized,
granules and
LHreceptor
The
complex
is
then
degraded and the LH receptor is recycled via secretory
incorporated
in
the
plasma
membrane
by
al.,
1972)
exocytosis
(Niswender et al., 1981).
Mechanism of Luteolysis
In
the
nonpregnant
(Robinson, 1977),
ewe
(Thorburn
et
and
cow
the corpus luteum regresses abruptly 13 to 15 days
and 18 days following ovulation,
respectively.
evidence that PGF2a of uterine origin is the
There is substantial
agent
responsible
for
14
normal regression of the corpus luteum in the ewe and cow (for review
see Goding, 1974;
Horton and Poyser, 1976;
Stormshak et al.,
1987).
decline
Corpus
Hansel and Convey, 1983;
regression
luteum
results
in
a
gland
(functional
luteolysis) followed by degenerative changes (structural
luteolysis)
of
the
secretory
of
activity
the
such as a decrease in cytoplasmic granulation, a rounding of the cell
outline
and peripheral vacuolation of the large luteal cells (Hansel
et al., 1973; Stormshak et al., 1987).
Functional
corpus
luteum
synthesis
and
luteolysis
to
PGF2a
culminates
and
secretion.
Prostaglandin
luteolysis by interfering with LH
activity
1975).
within
luteal
from short term exposure of the
results
plasma
1982),
bovine
of
(Marsh,
2
in
vitro
resulted
cyclase
and McNatty,
(Fletcher
and
human (Hamberger et al.,
1971),
1979) corpora lutea
to
gonadotropin induced
of
inhibition
in
ovine
1976),
1979) and nonhuman primate (Stouffer et al.,
PGF a
adenylate
(Henderson
membranes
progesterone
may induce functional
F2a
activation
Exposure of rat (Lahav et al.,
Niswender,
reduced
in
adenylate cyclase activity and cAMP production.
Furthermore, natural
luteolysis in cattle (Garverick et al.,
pigs
al.,
1986)
and
primates
(Eyster
1985),
et al.,
induced luteolysis in ewes (Agudo et al.,
rats (Khan and Rosberg,
(Ritzhaupt
et
1985) as well as PGF2a
1984)
and
pseudopregnant
1979) was accompanied by a decrease in basal
and(or) LHstimulated adenylate cyclase activity.
Prostaglandin F2a has also
phosphoinositide
metabolism
in
been
recently
shown
bovine (West et al.,
to
stimulate
1986) and rat
15
(Leung et al.,
resulted
1986) luteal cells in vitro.
generation
in
of
PGF2a
of
action
This
inositol 1,4,5triphosphate and diacyl
glycerol whose actions as second messengers in target tissues
elicit
calcium mobilization from the endoplasmic reticulum and activation of
Nishizuka
protein kinase C, respectively (Berridge and Irvine, 1984;
et al., 1984).
those
Moreover calcium effects on intact luteal cells resembled
of
Calcium
PGF2a.
ionophore
A23187
LHinduced
inhibited
cAMP
accumulation in rat luteal cells (Dorflinger et al., 1984) and caused
an
increase
inhibition
intracellular
in
LHstimulated
of
calcium
resulted
that
levels
in
progesterone synthesis by small bovine
luteal cells (Hansel and Dowd, 1986).
Elevated
intracellular
calcium levels may result in activation
An increase in the
of many enzymes that affect luteal cell function.
activity
phosphodiesterase,
of
inactivates
ewes (Agudo et
calcium
was
cAMP,
al.,
may
levels
phospholipase A
2
calciumdependent
In
addition,
increased
intracellular
decrease plasma membrane fluidity by activating
which catalyzes release
arachidonic
of
were suggested to result
Such
changes
from
could
elevations
accelerate
acid
in
luteal
luteal
2
the
action
activity
prostaglandin
regression in a
positive feedback manner and(or) the generation of superoxide
through
from
Degenerative changes in
membranes correlated with increased phospholipase A
synthesis.
that
2
phospholipid (Riley and Carlson, 1985,1986).
plasma
enzyme
within 2 h of PGF a administration to
noted
1984).
a
anions
of lipoxygenases on arachidonic acid (Riley and
16
Carlson,
uterine
1985).
or
Rothchild (1981) proposed
ovarian
origin,
whether
PGF2a
of
stimulate its own production in
could
luteal tissue of all species,
that
thus contributing to the completion of
luteolysis in a paracrine fashion.
Chronic
exposure
structural luteolysis.
listed
there
above,
to
PGF a
In
addition
was
more
for
2
to
than
24
morphological
the
activity
the
in
changes
decrease in LH receptor concentration,
a
membrane fluidity and steroidogenic enzyme activity
increase
results in
h
of
as
well
as
an
lysosomal enzymes (Stormshak et al.,
1987).
Prostaglandin
indirectly.
the
may
F2a
affect
directly
tissue
luteal
or
A direct action of PGF2a on luteal cells is supported by
existence
of
specific
receptors
membranes of ovine (Powell et al.,
located
1974a),
within
the
bovine (Powell
plasma
et
al.,
1976; Lin and Rao, 1977), equine (Kimball and Wyngarden, 1974), human
(Powell et al.,
lutea.
1974b) and rat (LuborskyMoore et al., 1979) corpora
Prostaglandin
indirectly
Fla
may
also
affect
corpus
the
luteum
by restricting the blood flow through the luteal vascular
bed but such an effect of PGF2a is
equivocal
(Nett
et
1976;
al.,
Niswender et al., 1976).
Endocrine Regulation of IIGF2(1 Secretion
Prostaglandins are secreted by the
(Thorburn et al.,
1973).
uterus
in
sporadic
pulses
Mean concentrations of PGF a in the utero
ovarian venous plasma are elevated between days
2
12
and
14
of
the
17
estrous
cycle
(Silvia
ewes
in
et
leading to luteal
1984)
al.,
regression and the beginning of a new cycle.
Endocrine regulation of PGF a secretion in ewes
2
controlled
1984).
primarily
by
According to
progesterone
permitting
synthesis
estradiol
appear
endogenous
the
regression
transfer of PGF
2a
is
from
estradiol
to
stimulate
of
oxytocin
receptor
Endogenous luteal oxytocin interacts
initiated
the
endometrium.
a result of the countercurrent
as
uterine
the
be
phase progresses,
luteal
with its receptor to cause secretion of PGF2a from
Luteal
actions
uterotropic
as
the endometrium.
in
the
decline
to
to
oxytocin (McCracken et al.
and
hypothesis
this
appears
vein
to
ovarian
the
artery.
Further release of oxytocin from the corpus luteum is caused by PGF2a
and
oxytocin
release
so
binding
that
the
to
the
two
endometrium further reinforces PGF2a
hormones
undergo
a
positive
feedback
interaction leading to complete luteolysis.
Embryonic Luteotropins
Maintenance of pregnancy requires extension of the life span and
function of the corpus luteum.
In the ewe it is
necessary
for
the
conceptus to be in the uterus by day 12 or 13 postestrus to exert its
antiluteolytic effect (Moor and Rowson,
referred to as
pregnancy.
the
Because
critical
the
embryo
for
This period is often
maternal
recognition
of
attachment of the trophoblast to the endometrium
in this species does not occur
1951),
period
1966).
until
day
18
postestrus
(Amoroso,
somehow prevents luteolysis 4 to 5 days prior to
18
attachment to the uterus (Niswender et al., 1985).
The primary mechanism by which the conceptus prevents luteolysis
is not
fully
uterine
understood.
venous
plasma
Measurement
revealed
Pexton et al.,
Silvia
et
1984).
al.,
effective
as
1975;
pregnant
in
of a
PGF a
cause
to
al.,
was
less
nonpregnant and
1976;
Moreover, the ability
superovulated
in
ewes was inversely related to the number of embryos present
in the uterus (Nancarrow et
indicate
PGF2a
in
regression
luteal
et
Mapletoft et al.,
Nancarrow et al., 1982).
analog
2
pregnant
1977;
in
Lewis et al., 1977;
than
hysterectomized ewes (Inskeep et al., 1975;
Pratt et al.,
(Wilson
exogenous
addition,
In
concentration
postestrus
Nett et al., 1976;
luteolysin
a
PGF2a
no difference between pregnant and
nonpregnant animals on comparable days
1972;
of
that
al.,
1982).
Collectively,
these
data
conceptus inhibits the PGF2a luteolytic activity
the
rather than suppresses its secretion.
Maintenance
of
domestic
animals
steroids
and
al., 1981;
synthesis
appears
proteins
(Stormshak et al.,
Anderson,
luteal
1970),
to
that
function
regulated
be
function
by
gestation
in
conceptusderived
primarily as antiluteolysins
Rate of uterine blood
1987).
flow
(Griess
and
uterine secretion of PGE2 (Marcus, 1981, Silvia et
LaCroix and Kann, 1982;
(Findlay
early
during
et al.,
Silvia et al., 1984) and protein
1981) all increase at the critical time
for maternal recognition of pregnancy.
Treatment
nonpregnant
of
ewes
ewes
(Pratt
with
PGE
et al.,
2
delayed
1977;
luteal
regression
Magness et al.,
in
1981) and
19
blocked the luteolytic action of PGF2a when the
simultaneously
administered
1977;
al.,
Reynolds
estradiolinduced
infusion of PGE
2
et
(Chenault,
prevented by intrauterine
in ewes (Pratt et al., 1977; Colcord et al.,
or PGE
1
slices
and
secreted
more
Endometrial
1983).
1978) and heifers
Huecksteadt and Weems,
preparations from pregnant
ewes
dissociated
LaCroix and Kann,
1982) and
PGE2
also
is
cell
than
similar
1979b;
Marcus
secreted
during
PGE2
preparations from nonpregnant ewes (Ellinwood et al.,
1981;
and
natural
addition,
In
are
regression
luteal
Hoyer et al., 1978;
1978;
1981).
al.,
Mapletoft et
1977;
(Henderson et al.,
were
compounds
two
incubation of ovine (Hyland et al., 1982; LaCroix and Kann, 1982) and
bovine (Shemesh et al., 1979; Lewis et al., 1982) blastocysts.
Prostaglandin
action
on
luteal
stimulating
E2
could maintain luteal function through direct
cells
adenylate
(Fletcher and Niswender,
binding
by
cyclase
to
by
and
receptors
and progesterone synthesis
activity
1982) or
specific
antagonizing
the
luteolytic
embryos
had
no
actions of PGF2a (Fitz et al., 1984).
Unlike
PGE2,
homogenates
stimulatory effect on secretion of
luteal
cells
directly
into
contrast
these
and
the
did
not
uterine
homogenates
ovine
of
prolong
vein
luteal
(Ellinwood
prolonged
et
al.,
1979a).
However,
cultured
function
et
al.,
ovine
when infused
1979a).
In
luteal function when infused
into the uterine lumen (Rowson and Moor, 1967;
Ellinwood
by
progesterone
direct
Martal et al.,
1979;
this luteotropic property was
destroyed by treatment with heat or proteolytic enzymes
(Rowson
and
20
Moor, 1967;
Martal et al., 1979) suggesting that the factor secreted
by the embryo, which ultimately resulted in maintenance of the corpus
luteum, was a protein.
Characterization of proteins secreted by day 13 ovine
revealed
three
closely
proteins that are now collectively
related
called ovine trophoblast protein 1
Secretion
of
occurred
oTP-1
gestation and appeared to
conceptuses
have
also
Godkin
(oTP-1;
shown
from
16-24 of gestation (Bartol et al.,
1985).
days
1982).
12-21
of
Bovine
group of acidic
a
proteins referred to as bovine trophoblast protein 1
days
al.,
trophoblast.
the
secrete
to
et
between
transiently
originate
been
conceptus
(bTP-1)
between
An additional group
of bovine conceptus proteins are also produced between days 21-38
of
gestation (Godkin and McGrew, 1986).
Infusion of total conceptus proteins,
nonpregnant
ewes
between
days
12-18
into the uterine lumen of
postestrus (Rowson and Moor,
1967; Martal et al., 1979) and introduction of bTP-1 into the uterine
lumen of nonpregnant cows
estrous
cycle.
In
(Thatcher
addition,
et
ability
al.,
1985)
and
ewes,
has
led
to
the
the
of trophoblastic vesicles to
prolong luteal maintenance after interspecies transfer
cows
prolonged
elegant
to
demonstration
recipient
of
the
immunological homology that exists between oTP-1 and bTP-1 (Helmer et
al., 1987).
Further characterizaion of these conceptus proteins will
likely enhance our understanding of
their
underlying
mechanism
of
action in promoting luteal maintenance during early gestation in ewes
and cows.
21
Paracrine Regulation of Luteal Function
recently,
Until
corpus luteum to LH and
increasing
evidence
dogma
central
demise
its
that
luteal
to
function
but
there
However,
PGF2a.
controlled not only by secretions from the
uterus,
maintenance of the
attributed
domestic animals is
in
adenohypophysis
also by secretions from within the ovary.
the
corpus
luteum
of
species
several
the
composed
is
the
and
Evidence for
paracrine regulation of luteal function is strengthened by
that
is
fact
of two
different cell types (Mossman and Duke, 1973); one of which is devoid
of LH receptors and does not respond to this hormone, while the other
is unresponsive to PGF2a because it lacks receptors for this compound
(Niswender et al.,
variety
of
1985).
protein
and
In addition one type of cell
peptide
metabolism of the other cell type and
luteum
in
general.
hormones
the
may
that
function
of
secretes
affect
the
a
the
corpus
Current concepts on the paracrine regulation of
luteal function will be reviewed in this section.
Cell Types of the Corpus Luteum
As early as 1919, Corner reported that the porcine corpus luteum
was composed of two cell types.
yet
Significance
of
this
interesting,
simple observation was not recognized until recently when it was
discovered that porcine (Lemon and Loir,
1977),
bovine (Ursely
and
Leymarie, 1979; Koos and Hansel, 1981; Alila and Hansel, 1984), ovine
(Rodgers and O'Shea, 1982;
rabbit (Hoyer et al.,
Fitz et al., 1982;
O'Shea et al., 1986),
1986) and primate (HildPatito et
al.,
1986)
22
corpora
lutea consist of small (12-22 um) and large (22-40 um) cells
that are morphologically and biochemically different.
is
characterized
by
a
spindle,
lamina surrounding large luteal cells is
large luteal cells is
characterized
microvillous folds (Enders,
structural features
by
of
that
than
prominent
1979) and the surface of
presence
the
of
numerous
Small and large cells share fine
1973).
typical
In contrast the basal
more
associated with small cells (O'Shea et al.,
darkstaining
shape,
elongated
and an irregularlyshaped nucleus.
cytoplasm,
The small cell
steroid
producing
namely,
cells,
numerous mitochondria, abundance of smooth endoplasmic reticulum, and
large
lipid
droplets.
contrast
In
only
the
large
luteal cells
possess the cytoplasmic machinery characteristic of cells specialized
for polypeptide and protein secretion.
complexes,
This includes numerous
Golgi
extensive rough endoplasmic reticulum, and electron dense
secretory granules (Niswender et al., 1985).
In addition to these morphological
the
small
and large cells differ in several biochemical parameters.
There are numerous receptors for LH on small luteal
few
large cells.
on
for PGF a and PGE2,
Moreover,
Conversely,
estradiol
but
cells
very
the large cells contain receptors
whereas the small cells do
2
1982).
differences
functional
and
not
(Fitz
et
al.,
receptors are fivefold more abundant in
large luteal cells (Glass et al., 1985).
Regulation of steroidogenesis in the two cell types
be
quite
different.
In
particular,
regulating steroid secretion
by
large
the
mechanisms
cells
are
not
appears
to
involved
in
clear.
The
23
observation
that
secretion of progesterone in small
stimulates
LH
but has no effect on large cells
cells,
because
large
cells
secrete
important
particularly
is
approximately
times
20
much
as
progesterone as do small cells in the unstimulated state. Large cells
also account for approximately 30% of the corpus luteum on
basis
compared
with
a
volume
16% for small cells (Niswender et al.,
1985).
This becomes puzzling if LH
hormone.
cells
However,
into
possibility
is
developed
(Donaldson
cells
supported
(1984) and Niswender
against
to
be
luteotropic
the
may stimulate the transformation of the small
LH
large
considered
is
et
by
and
(1985).
al.
data of Alila and Hansel
recent
the
This
1965a).
Hansel,
Using
antibodies
monoclonal
theca and granulosaspecific antigens,
Alila and
Hansel (1984) examined the origin of small and large luteal cells
the
bovine
corpus
Early in the cycle,
antibody,
luteum
the estrous cycle and gestation.
during
on days 4-6,
of
70% of
small
cells
bound
while 77% of large cells bound granulosa antibody.
theca
As the
cycle progressed, on days 16-18, the number of large cells that bound
granulosa antibody decreased to only 30% while
theca
bound
antibody.
After
bound granulosa antibody.
the
day
40%
of
100 of gestation,
cells
no large cell
These data indicate a gradual increase
in
percentage contribution of small cells derived from theca origin
to the large cell population as the corpus luteum ages.
al.
large
(1985) treated ewes
with
human
chorionic
Niswender et
gonadotropin
(hCG)
during
midcycle
cells.
If LH transforms small cells into large cells, then it may be
and
reported
an
increase
in the number of large
24
possible that the machinery for progesterone synthesis is "locked on"
indefinitely during this transformation process.
Only if this is the
case could LH be the stimulus for progesterone synthesis by the large
cells.
Large luteal cells contain receptors for PGF2a and PGE2
small
cells
do
not (Fitz et al.,
whereas
Therefore the luteolytic
1982).
effects of PGF a on both large and small cells appear to be
mediated
2
by a cytotoxic or inhibitory factor(s) secreted by large luteal cells
(Niswender
et
1985).
al.,
another example for
provide
may
This
paracrine interaction between the large and small
luteal
cells
that
The
prostanoids
some
balance
between
et
The
large
PGE1 and prostacyclin to
are also stimulated by PGE2,
secrete progesterone in vitro (Fitz
cells.
1984).
al.,
This
suggests
are luteotropic while others are luteolytic.
these
chemical
species
is
for
essential
regulation of luteal function (KhanDawood and Dawood, 1986).
Large
cells
may
more
be
to estradiol because its
sensitive
receptors are fivefold more abundant than that of small cells
(Glass
et al., 1985).
Although both small and
large
cells
secrete
a
large
variety
of
steroidogenic,
are
cells
only
and peptide hormones.
protein
Relaxin (Anderson and Long, 1978; Fields et al., 1980; Fields, 1984),
oxytocin (Wathes et al.,
1983a;
Thorburn,
and Fields,
Fields
1985;
vasopressin (Wathes et
corpora
lutea
of
al.,
Rodgers
1983a)
et
1986;
have
al.,
1983;
KhanDawood,
been
domestic animals and primates.
identified
Rice
and
1986) and
in
the
Among the peptide
25
hormones produced by the large luteal cell, oxytocin has received the
most attention.
Oxytocin concentrations increase
tissue
luteal
in
and in the general circulation during the luteal phase of the estrous
cycle
(Wathes
et
At
1986).
al.,
enhanced basal steroidogenesis in bovine and
while
cells,
higher
at
concentrations
production and the response of
1982a,b).
Similarly,
cells
may
luteal
inhibited progesterone
it
cells
cultured
human
hCG
to
(Tan
et
al.,
inhibited LHstimulated progesterone
oxytocin
secretion from cultured small
small
these
oxytocin
concentrations,
low
ovine
luteal
cells
that
indicating
have oxytocin receptors (Niswender et al.,
These observations suggest that oxytocin may serve as an
1985).
intraluteal
communicator between large and small cells.
Aten
et
(1986a,b)
al.
reported
the
presence of a GnRHlike
ovarian hormone (GLOH) in rat and human ovaries and proposed that
it
may be the physiological ligand that binds to the "GnRH" receptors in
ovary
the
because
it
radioreceptor assay.
role
showed
substantial
activity
in
Thus this ovarian protein may play a
a
GnRH
paracrine
by inhibiting luteal function if its effects on the ovary would
resemble those of GnRH (see GnRh section).
Extracts of the sheep, goat, pig, dog, cat, rat and human corpus
luteum have been shown
binding
to
inhibitor
1981).
ages.
its
to
contain
a
substance
Kumari et al., 1980;
Ward
inhibits
LH
LHreceptor binding
receptor and is referred to as
(LHRBI;
that
,
1981;
Yang et al.,
The LHRBI concentration increases in the corpus luteum as it
This suggests that LHRBI may regulate luteal function locally
26
by
inducing
luteolysis
through
inhibition
LH/hCG binding and
of
therefore progesterone biosynthesis in the late luteal phase.
that
Mechanisms controlling luteal function may involve factors
produced
are
both
within
the corpus luteum and outside the ovary.
These mechanisms involve a series
peptides,
steroids
and
of
molecular
prostaglandins,
each
species,
proteins,
may
which
of
act
independently or in concert modifying the actions of one another.
Synthesis and Secretion of Peptide Hormones by the Corpus Luteum
protein
The corpus luteum synthesizes and secretes a variety of
peptide hormones.
and
Relaxin (Anderson and Long,
al., 1980; Fields, 1984), oxytocin (Wathes et al., 1983a;
al., 1983;
Fields et
1978;
Rodgers et
Dawood, 1986), GnRHlike ovarian hormone (GLOH; Aten et al.
vasopressin
and
domestic
granules
animals and primates.
1986a,b)
1983a) have been identified in the
(Wathes et al.,
electrondense secretory
Khan
Fields and Fields, 1986;
Rice and Thorburn, 1985;
of
the
large
luteal
cells
of
Synthesis and secretion of oxytocin,
GLOH and relaxin from the corpus luteum as well as their actions will
be discussed.
Oxytocin in The Corpus Luteum
A corpus luteum factor with oxytocic
early
as
the beginning of this century.
action
was
suggested
as
Ott and Scott (1910) found
that an aqueous extract of corpus luteum increased milk flow
in
the
27
Schafer
and
goat,
and Mackenzie (1911) and Mackenzie (1911) showed
that injection of an extract of ovine corpus luteum induced milk let
down in the lactating cat.
pursued
further
These interesting observations
this
until
corpora lutea of several
(Fields et al., 1983;
and
not
decade when oxytocin was identified in
corpus
The
species.
luteum
of
cow
the
Wathes et al., 1983a,b) ewe (Wathes and Swann,
1982) monkey (KhanDawood et al.,
KhanDawood
were
1983)
Dawood,
1984) woman (Wathes et al.,
1982;
and rabbit (KhanDawood and Dawood,
1984) has been shown to contain measurable quantities of oxytocin.
Comparison of Luteal and Hypothalamic Oxytocin
Luteal oxytocin is not only
but
is
also
biologically
Luteal extracts caused
immunologically
similar
pituitary counterpart.
increase
significant
a
its
to
indistinguishable,
contraction
in
of
uterine muscle, stimulated contraction of uterine strips in vitro and
increased
intramammary
pressure
oxytocin in rats (Wathes and Swann,
Moreover
ovine,
bovine
in
a
1982;
manner similar to authentic
Wathes
et
al.,
1983b).
and human luteal oxytocin extracts elute at
the same position as pituitary oxytocin by Sephadex G50 and
reverse
phase highperformance liquid chromatography (HPLC; Wathes and Swann,
1982; Wathes et al., 1982; Wathes et al., 1983a; Fields et al., 1983;
Sheldrick and Flint, 1983a;
Dawood,
1986).
Schaeffer et al., 1984; Dawood and Khan
Dispersed cell cultures of ovine and bovine
corpora
lutea incorporated labeled cysteine into a peptide that eluted at the
28
same
position
the synthesis
As occurs in the hypothalamus,
oxytocin on HPLC.
as
of
oxytocin
luteal
involved
the
of
formation
an
approximately 14K precursor protein that was subsequently cleaved to
form oxytocin and neurophysin (Swann et al.,1984).
is
transcribed
highly
in
sequence analysis as well as
that
luteal
similar.
250
more
mRNA
Luteal cDNA
corpus luteum.
bovine
cellfree
hypothalamic
and
However,
times
the
The oxytocin gene
showed
studies
translation
oxytocin were essentially
for
the active corpus luteum
approximately
produces
oxytocin mRNA than a single hypothalamus (Ivell and
Richter, 1984).
Variations in Luteal Oxytocin Levels
In the ewe and cow oxytocin is first
detectable
in
follicular
fluid and granulosa cells during or shortly after the LH surge (Kruip
et al., 1985;
Wathes et al., 1986).
Measurement of luteal oxytocin
specific mRNA throughout the estrous cycle of
gene
transcription
these
cow
showed
that
was maximal accompanying ovulation and decreased
Maximal concentrations of mRNA were detected around
thereafter.
3;
the
day
declined sharply around day 7 and reached a basal level by
day 11 of the cycle, after which only very low levels were detectable
(Ivell et al.,
1985).
Measurement of oxytocin mRNA
levels
in
the
bovine hypothalamus revealed no significant variation due to stage of
the
estrous
cycle
(Ivell
et
al.,
1985).
This
implies that the
factors that regulate transcription of the oxytocin gene are
tissue
29
specific
and
the hypothalamic gene is regulated independently
that
from its luteal counterpart (Ivell, 1986).
Changes in luteal concentrations of oxytocin during
estrous
cycle
tissues
luteal
do not occur simultaneously with those of
apparently
mRNA for this peptide.
Oxytocin concentrations in ovine
have
been
In
the
luteal
cow,
bovine
and
shown to be maximal at day 8 and between
days 5-10, respectively (Sheldrick and Flint,
1984).
bovine
the
Wathes et al.,
1983b;
oxytocin concentrations increased from
about .5 pg/g (wet weight) on days 1-4 of the cycle to more than
pgig
days
on
and declined thereafter to about 1 pg/g between
5-10
days 11-17 and to less than .6 pg/g after
1984).
similar
A
day
(Wathes
18
et
al.,
pattern was observed for the ewe by Sheldrick and
Flint (1983b) who
found
maximal
oxytocin
occurrence
maximal
of
concentration
in
ovine
The observed difference in time
luteal tissue on day 8 of the cycle.
between
1.7
levels
of
mRNA
oxytocin
and
of
concentrations of this peptide hormone in luteal tissue suggests that
during early stages of the cycle
processed.
This
lag
period
only
some
of
prohormone
the
is
may be controlled by certain endocrine
factors that regulate synthesis and
processing
of
the
hormone
in
measured
in
granulosa cells (Wathes et al., 1986).
In contrast to the
ruminants,
extremely
high
oxytocin
concentrations
low concentations were found in the sow corpus
luteum,
with maximal levels (10 ng/g) detected on day 5 of the cycle
(Pitzel
et al.,
1984).
Similarly,
both rat and rabbit ovarian and
30
luteal tissues, respectively, contained low levels of oxytocin (Khan
Dawood and Dawood,
tissue
was
also
Oxytocin concentration in primate
1984).
much
lower than that of ruminants.
(1982) detected oxytocin levels of about 30
and
Dawood
(1983)
human luteal tissue.
from
34
to
602
Wathes et al.
KhanDawood
while
concentrations of 59 ng/g in
oxytocin
reported
ng/g
luteal
ranged
In cynomolgus monkeys the concentration
ng/g
wet
weight
of
concentration at the midluteal phase of
tissue,
with
cycle
the
highest
the
(KhanDawood
et
cells
is
al., 1984).
Oxytocin
is
produced
by
the
large
luteal
and
temporarily stored in membranebounded secretory granules (Rodgers et
al., 1983;
Fields,
Rice and Thorburn, 1985;
Fields and
Kruip et al., 1985;
This peptide hormone is secreted into ovarian
1986).
of cows (Walters et al.,
veins
1984) and ewes (Flint and Sheldrick, 1982),
thereby contributing to the relatively high levels of oxytocin in the
systemic circulation of these species during the luteal phase of
the
estrous cycle (Webb et al., 1981; Sheldrick and Flint, 1981; Mitchell
et al.,
1982;
Schams et al.,
1982;
Schams, 1983;
1984; Walters and Schallenberger, 1984).
phase in both species is characterized by
In contrast, the follicular
low
concentrations that decline to basal levels at
(Wathes
et
al.,
1986).
This
decline
Walters et al.,
in
the
time
of
estrus
oxytocin concentrations
either preceded or occurred simultaneously with that of
(Schams, 1983; Flint and Sheldrick, 1983).
oxytocin
circulating
progesterone
31
Control of Luteal Oxytocin Secretion
As discussed in a
previous
review,
ovarian
secretion of oxytocin is believed to be stimulated by PGF2a.
Indeed,
treatment
of
cows
and
ewes
immediate increase in luteal
Flint and Sheldrick,
1983;
with
PGF2astimulated
secretion
PGF2a caused an
(Walters
et
al.,
1984) and
Evidence
1983).
al.,
for
release of ovarian oxytocin is provided
in part by the close relationship that exists between the
and
et
Schallenberger et al.,
1983;
degranulation of luteal cells (Heath
endogenous
of
analog
an
oxytocin
this
of
section
occurrence
frequency of episodic secretion of this ovarian hormone and that
of 13,14dihydro-15ketoPGF2a,
and Sheldrick,
release of PGF
1982).
2a
a stable metabolite of PGF2a
(Flint
Conversely, oxytocin treatment stimulated the
from the uterine endometrium of the ewe (Roberts and
McCracken, 1976), goat, (Cooke and Homeida, 1982) and cow (Newcomb et
al., 1979; Milvae and Hansel, 1980).
Actions of Ovarian Oxytocin
The
first
evidence
described by Armstrong
(1960).
Injection
cycle resulted in a
cycle.
These
al., 1964;
data
of
and
of
a
effect
luteolytic
Hansel
(1959)
and
of oxytocin was
Hansel
and
Wagner
oxytocin into heifers between days 3-6 of the
significant
decrease
were
confirmed in the cow (Labhsetwar et
later
Donaldson and Takken, 1968;
(Cooke and Knifton, 1981;
in
duration
of
estrous
Harms et al., 1969) and goat
Cooke and Homeida, 1982).
It appears that
32
the
luteolytic
action
of oxytocin is normally mediated by PGF2a as
hysterectomy
explained above because it can be prevented by
cow (Armstrong and Hansel,
1967) and by simultaneous inhibition of prostaglandin synthesis
al.,
in
Ginther et
Anderson et al., 1965;
1959;
the
in
the
goat
Further,
(Cooke and Knifton,
both
active
and
Cooke and Homeida,
1981;
passive
against
immunization
1983).
oxytocin
extended the length of the cycle in ewes and goats (Sheldrick et al.,
1980;
Schams et al.,
rhesus monkey,
oxytocin
has
1983;
mare,
sow,
Cooke and Homedia,
1985).
In the ewe,
rabbit and guinea pig injection of
rat,
far proved ineffective in reducing estrous cycle
thus
length (Duncan et al., 1961;
Donovan, 1961;
Brinkley and Nalbandov,
1963; Milne, 1963; Neely et al., 1979; Wilks, 1983).
Oxytocin may also have a direct effect
regulation
of
luteal
stimulated progesterone
luteal
function.
low
In
production
on
steroidogenesis
concentrations,
isolated
by
cells in the early luteal phase,
bovine
whereas,
et
al.,
1982a,b).
This
latter
effect
that
oxytocin
cells to hCG in vitro.
inhibited
the
and
human
concentrations
of
progesterone production was confirmed by Niswender et al.
found
oxytocin
it inhibited both
basal and hCGstimulated progesterone release at high
(Tan
and
oxytocin
on
(1985) who
response of small ovine luteal
However, numerous studies conducted by others
have failed to confirm that oxytocin can act directly at the level of
the corpus
luteum
in
human
(Richardson
and
Masson,
1985),
rat
(Mukhopadhyay et al., 1984) and cow (Wathes et al., 1986) to suppress
33
progesterone
Nevertheless,
secretion.
using rat testicular cells in
culture,
and Hsueh (1981a,b),
Adashi
have
demonstrated
a
dose
dependent inhibitory effect of oxytocin, vasopressin and vasotocin on
testosterone secretion.
was
due
It was later shown that the vasotocin effect
inhibition
to
of
desmolase that convert progesterone to
Hsueh,
1982).
luteum is
A
likely
similar
(Adashi
androstenedione
and
of action of oxytocin in the corpus
site
inhibit
to
17ahydroxylase and 17-20
enzymes
the
androgen
and
rather
estrogen
than
progesterone production by the ovary (Wathes et al., 1986).
A
role
for oxytocin in regulating steroidogenesis is supported
by the recent findings
that
oxytocin
steroidogenic
such
as
tissues
testis (Fields et al., 1983;
1984;
Schams
et
also
Makino et al., 1983;
and
Jayasena,
in
placenta,
other
and
Nicholson et al.,
Concentration of placental oxytocin
increased during the second trimester and remained
(Lederis
produced
the adrenal cortex,
1985a).
al.,
is
1970).
In
the testis,
high
until
term
immunocytochemical
studies have shown that 80% of interstitial cells stain for oxytocin.
This
suggests
that
(Guldenaar
and
testis may
stimulate
oxytocin
Pickering,
is
1985).
uterine
and
produced
Oxytocin
tubular
by
the
Leydig
cells
both
placenta and
motility,
respectively
in
(Wathes et al., 1986).
GnRHLike Ovarian Hormone
Gonadotropin releasing hormone (GnRH) of hypothalamic origin was
34
thought to act exclusively on the pituitary gland to increase release
of
LH
and
However an extrapituitary action of this releasing
FSH.
hormone was suggested by Rippel and Johnson (1976)
the
inhibitory
effect
demonstrated
a GnRHagonist on hCGstimulated augmen
of
tation of ovarian and uterine weights in
immature
hypophysectomized
inhibitory effects of GnRH or
Since this initial observation,
rats.
who
its agonistic analogs on luteal function
been
has
demonstrated
in
many species including the rat (Kledzik et al., 1978; Harwood et al.,
Jones and Hsueh, 1980), human (Koyama et al., 1978; Casper and
1980;
Yen, 1979), monkey (Asch et al., 1981) and cow (Rodger and Stormshak,
1986).
Exogenous GnRH or GnRHagonistic analogs may act directly on the
ovary to alter steroidogenesis.
granulosa
with
cells
Treatment of primary cultures of rat
GnRH or its agonists inhibited FSHstimulated
estrogen and progestin production (Hsueh and
1979;
Erickson,
Hsueh
and Ling,
1979),
and FSHstimulated LH and prolactin (PRL) receptor
formation
(Hsueh
and
inhibitory
effects
of
Ling,
GnRH
1979;
on
Hsueh
granulosa
et
1980).
al.,
cells
were
concomitant treatment with a GnRH antagonist (Hsueh and
Hsueh
et al.,
1980).
These
blocked by
Ling,
1979;
In addition to suppression of steroidogenesis
and receptor formation, GnRH has been shown to inhibit FSHstimulated
cAMP formation (Clark et al., 1980; Knecht et al., 1981), and enhance
FSHstimulated prostaglandin production by rat granulosa cells (Clark
et al.,
1980).
Treatment
with
GnRH
also
inhibited
progesterone
35
production
stimulated by hCG,
epinephrine,
or LH during short term
incubation of rat luteal cells (Clayton et al., 1979; Harwood et al.,
1980;
Behrman et al.,
Massicotte
1980;
et
al.,
However,
1981).
higher levels of LH and hCG alleviated the inhibitory effects of GnRH
on
progesterone
production
the rat (Behrman et al.,
in
1980) and
human (Casper et al., 1980), respectively.
Although
the rat model has been used extensively to examine the
effects of GnRH on ovarian functions,
direct effects of GnRH are not
limited to this species. In vitro treatment with GnRH or its agonists
modulates
steroidogenesis
in ovarian cells from pigs (Massicotte et
al., 1981) and chickens (Takats and Hertelendy,
al.,
1982).
1982;
Hertelendy et
contrast no direct inhibition of steroidogenesis by
In
GnRH was found in studies using ovarian tissues of
mice
and
rhesus
monkeys (Asch et al., 1981; Bex et al., 1982)
The bovine corpus luteum may be more sensitive to GnRH treatment
during the mid than
intravenous
early
injection
luteal
GnRH
of
phase
On
the
other hand,
the
cycle.
A
single
into cows on day 2 of the cycle was
followed by a 6 day lag period before
detected.
of
altered
luteal
function
was
a similar injection on day 10 of the
cycle caused serum concentrations of progesterone to be significantly
depressed after
investigators
only
have
48
h
(Rodger
and
Stormshak,
1986).
Other
shown that injection of GnRH or a GnRH agonistic
analog into cows during the midluteal phase of
cycle
increased
serum concentrations of progesterone (Kittok et al., 1973;
Milvae et
the
36
1984).
al.,
function
However,
of
(1984)
Milvae et al.
GnRHtreated
reported
that
luteal
cows was suppressed during the succeeding
cycle.
Several attempts have been made to elucidate the mechanism(s) by
which GnRH affects ovarian function.
that
(1980) suggested
Casper et al.
GnRHinduced early luteal regression in the human may be due to
a decrease in gonadotropin secretion caused by GnRHinduced pituitary
refractoriness.
may
have
of
It has also been proposed that the inhibitory
1986).
GnRH
rat
in
luteal
results from ability of the
cells
decapeptide to inhibit calcium extrusion from the
and Behrman,
1983).
Consequently,
Cdependent
(Williams
cytosol
elevated levels of intracellular
calcium prevent activation of adenylate cyclase by LH via
kinase
that
resulted in downregulation of luteal LH receptors (Rodger
and Stormshak,
action
In the cow GnRH injection caused release of LH
a
protein
phosphorylation or calmodulindependent process.
This mechanism of action of GnRH is supported by recent data of Leung
et al.
(1986) who found that GnRH binding
receptors
to
in
plasma
membranes of rat luteal cells in primary culture activated hydrolysis
of
phosphoinositides,
which
ultimately
leads
to
stimulation
of
protein kinase C activity.
To demonstrate the
ovarian
actions
of
physiological
GnRH,
significance
the
direct
it was deemed necessary to establish the
presence of a GnRHlike substance as well as
ovary.
of
its
receptors
in
the
The presence of such a molecule would explain the paradoxical
37
inhibitory
effect of this releasing peptide on ovarian functions and
provide unique models for understanding the mechanism of GnRH
on steroidogenesis and ovulation (Hsueh et al.,
Gonadotropin
1984).
releasing hormone receptors have been detected in
rat
action
cells
luteal
(Clayton et al., 1979), but only low affinity binding sites have been
found in the human
Popkin et al.,
corpus
1983).
luteum
(Clayton
Huhtaniemi,
and
1982;
However, no GnRH receptors have been detected
in bovine, ovine and porcine luteal tissue (Brown and Reeves,
Although
several
reports
suggested
have
that GnRH or a GnRHlike
peptide is synthesized in the ovary and exerts
(Williams and Behrman, 1983;
its
Birnbaumer et al., 1985), only recently
rat luteal tissues (Aten et al.,
was found to contain a
locally
effects
has the presence of such a molecule been demonstrated in
and
1983).
GnRHlike
1986a,b).
protein
that
both
human
Rat ovarian extract
exibited
the
same
membrane binding properties as GnRH but was distinctly different from
the
hypothalamic
decapeptide
in
several
respects.
Although this
ovarian protein showed substantial activity in the GnRH radioreceptor
assay,
GnRH
it was immunologically refractory to a sensitive and specific
antiserum
Although,
both
and
exhibited only little immunoassayable activity.
radioreceptor
and
immunological
activities
were
detected in the hypothalamus, both activities were absent from plasma
extracts of the same animals.
In addition, the GnRHlike activity of
the
ovarian extracts was sensitive to elevated temperatures that did
not
affect
its
hypothalamic
counterpart.
Gonadotropin
releasing
38
hormonelike
activity
significantly
was
reduced
either 50 or 60°C for as little as 5 min,
by
incubation
activity
of
fractionated
authentic
ovarian
when
by
extracts
did
phase
ovarian
GnRH
like
when
The peaks did not elute with
extracts
GnRH
containing
an identical procedure (Aten et al.,
ovarian
affected
the GnRHlike
Moreover,
behave
not
HPLC.
subsequent experiment Aten et al.
GnRHlike
but GnRH was not
for up to 60 min.
reverse
by
GnRH
fractionated
60°C
at
by incubation at
1986b).
were
In a
(1986a) reported the presence of a
hormone (GLOH) in the human ovary.
This molecule
They proposed
was similar if not identical to GLOH of the rat.
that
GLOH may be the physiological ligand that binds to the socalled GnRH
receptors in the rat ovary and speculated that receptors for GLOH may
be
present
in human ovaries as well.
of GLOH in control of
ovarian
Knowledge of the significance
function
in
human
rat,
and
other
species awaits the purification of this protein.
Relaxin in the Corpus luteum
Evidence
for
a
unique
principle in aqueous extracts of swine
corpora lutea that relaxed the guinea pig pubic symphysis
presented
by
identified in
Hisaw
ovaries
(1926,
of
1927).
the
pig
Since
then
(Sherwood
relaxin
and
was
has
O'Byrne,
first
been
1974;
Matsumoto and Chamley, 1980; Fields et al., 1982), rat (Niall et al.,
1982),
cow (Fields et al., 1980;
Fields et al., 1982), human (Weiss
et al., 1976; O'Byrne et al., 1978) and monkey (Weiss et al., 1981).
39
Relaxins are species specific peptides with molecular weights of
just
under
6000
peptide chains,
daltons.
A and B,
They
composed
are
of two nonidentical
covalently linked by two disulfide bridges
with an additional intrachain disulfide bridge in the smaller A chain
(John
et al.,
Relaxin is derived from a larger precursor in
1981).
which A and B chains are linked by a connecting C peptide
The
1982).
al.,
hormone
insulin
resembles
in
(Niall
et
tertiary
size,
structure and mechanisms of cleavage from the primary RNA transcripts
(Schwabe et al., 1978).
and
insulin
are
This has led to the speculation that relaxin
derived
from
gene
ancient
an
underwent
that
duplication and modification. However, this concept has been recently
challenged
the
on
basis
of
very
homology in amino acid
limited
sequence between the two hormones (Schwabe et al., 1982).
Although
relaxin
concentrations,
is
during the estrous cycle in low
produced
the primary source of this
luteum of pregnancy (Anderson et al.,
1982).
suggested that relaxin is present in the
plasma
during
extraction,
biological
corpus
the
Wada and Yuhara (1961)
after
late
gel
Relaxin was isolated from
pregnancy
filtration,
activity
was
by
observing
that
and
major
active
isoelectric
determined
fractions
bovine
corpora
procedures including acidacetone
motility inhibition and by the mouse
Three
cow
is
from pregnant cows provoked a positive response in the guinea
pig bioassay for relaxin.
lutea
hormone
were
focusing,
and
its
by the assay of mouse uterine
interpubic
ligament
bioassay.
obtained with relaxin activity
40
chromatographed in the ranges of 1400 and 6,000
1980).
al.,
However,
isolation
presence
and
(Fields
et
of relaxin from
purification
and
bovine corpora lutea were complicated by the
relaxin
daltons
concentrations
low
of
of a factor that increased contraction of the
uterus in the mouse uterus bioassay that led to inconsistent measures
of relaxin biological activity (Fields et
al.,
factor
This
1980).
was later determined to be oxytocin (see oxytocin section).
Unlike
bovine
characterized by
levels.
luteal
low
tissue,
oxytocin
its
counterpart
porcine
concentrations
higher
and
relaxin
Therefore the pig is the best studied model for relaxin.
sows and gilts relaxin was first detected in follicular fluid,
contains a wide
1980).
range
Levels
of
concentrations
of
this
hormone
throughout the estrous cycle.
by
granulosa
granules.
lutein
cells
sow
in
(Matsumoto
and
which
Chamley,
relaxin is produced
stored in electrondense secretory
Number of relaxin containinggranules
throughout
In
tissue remain low
luteal
In pregnant sows,
and
is
increases
steadily
pregnancy to become maximal by days 105-110 of gestation.
By the day preceding delivery, the number of these granules begins to
decline and within 6 h of parturition all granulosa lutein cells
nearly
devoid
of
these
pattern agrees very well
Systemic
plasma
granules.
with
delivery
relaxin
secretion
1982).
from
the
This
ovary.
concentrations of porcine relaxin remain low during
the first 100 days of gestation,
before
(Anderson et al.,
are
and
rise
within
increase gradually
during
the
next
2
days
3
days
to
peak
41
concentrations 22 h before parturition (Sherwood et al., 1975).
that
it
was
impression
the
When relaxin was first discovered,
gained
functioned primarily to prepare the birth canal for passage
of term fetuses.
Exogenous relaxin elicited marked dilatation in the
with
pretreated
sows
uterine cervix in ovariectomized heifers
and
diethylstilbestrol (Zarrow et al., 1956;
Smith and Nalbandov, 1958).
This was
et
confirmed
later
Anderson
by
who
(1982)
al.
found
exogenous porcine relaxin to induce cervical dilatation at a stage of
gestation when endogenous blood levels of estrogens reach peak values
and
levels
progesterone
rats,
begin
to decline.
onehalf
cervical dilatation during the latter
coincides
increasing
with
immunoreactive relaxin
cervical
softening
(Steinetz
et
gestation
concentrations
of
addition
to
1980).
al.,
In
relaxin induces pubic symphysis
and dilatation,
glands
(Schwabe
et
al.,
implicated in the separation of the
weakening
blood
of
inhibits myometrial activity and affects the vagina
relaxation,
mammary
systemic
hamsters and
In mice,
of
insulinlike regulator of
reproductive
placenta
the fetal membranes (Weiss,
tissues.
carbohydrate
Purified
and
has
Relaxin
1978).
the
from
also
uterus
and
been
and
and may act as an
1981),
protein
metabolism
in
porcine relaxin stimulated glycogen
deposition, tissue growth and protein synthesis as indicated by amino
acid incorporation in uteri of
either
unprimed
ovariectomized rats (Frieden et al., 1982).
or
estrogenprimed
42
STATEMENT OF THE PROBLEM
In order to maximize the
animals,
efforts
have
reproductive
been
made
corpus
implicated
of
the
regulation
of
domestic
to develop effective methods for
regulating the life span of the
in
efficiency
the
luteum.
estrous
Oxytocin
cycle
has
been
when it was
demonstrated that treatment of cows with this nonapeptide resulted in
shorter cycles.
by
large
It was later demonstrated that oxytocin is
cells
produced
of the bovine and ovine corpus luteum and stored in
secretory granules, but factors controlling its synthesis and release
are poorly understood.
Oxytocin
concentrations
in
bovine
luteal tissue at different
stages of the estrous cycle are not well defined.
However,
oxytocin
and progesterone secretions appear to change concomitantly during the
cycle.
Moreover, in vivo ovarian secretion of oxytocin is stimulated
by PGF2a in both cows and ewes.
Research
presented
in
this thesis was undertaken to determine
oxytocin concentrations in luteal tissue at different stages
estrous
cycle
cycloheximide,
and release.
and
to
examine in vitro effects of PGF2a,
colchicine,
of
the
PGE2 LH,
and cytochalasin B on oxytocin synthesis
43
EXPERIMENTS 1 AND 2: PROSTAGLANDIN F2a INDUCED RELEASE OF
OXYTOCIN FROM BOVINE CORPORA LUTEA IN VITRO
INTRODUCTION
Oxytocin
has
Wathes et al.,
identified in bovine (Fields et al.,
been
1983a,b) and ovine (Wathes and Swann,
1983;
corpora
1982)
lutea where it is temporarily stored in granular form in large luteal
cells
(Fields
and Fields,
This peptide hormone is secreted
1986).
into ovarian veins of cows (Walters et al., 1984) and ewes (Flint and
Sheldrick,
oxytocin
1982),
in
thereby contributing to relatively high levels of
systemic
the
of these species during the
circulation
luteal phase of the estrous cycle (Webb et al.,
Flint,
Schams,
1981;
Changes
1983).
1981;
Sheldrick and
oxytocin
luteal
in
defined
concentrations during the bovine estrous cycle are not
well
because
dated
available
data
are
based
subjectively
on
tissue
acquired at the abattoir (Wathes et al., 1984; Schams et al., 1985a).
Ovarian secretion of oxytocin is stimulated by PGF2a.
of cows with a PGF2a analog caused an immediate
oxytocin
1984) and
secretion
(Walters
degranulation
of
et al.,
in
luteal
Schallenberger et al.,
1983;
cells
luteal
increase
Treatment
(Heath
et
al.,
1983).
Administration of the same analog to ewes (Flint and Sheldrick, 1983)
has
also
been
shown
to
result
in
a
significant
venoarterial
difference in oxytocin levels in ovaries bearing corpora lutea
addition,
a
transient
increase
in
systemic
.
In
plasma oxytocin that
44
occurred in PGF
treated intact ewes was absent in similarly treated
ovariectomized
release
ovarian
of
Evidence
ewes.
oxytocin
for
provided
is
relationship that exists between
the
PGF2astimulated
endogenous
part
in
occurrence
by
the close
frequency
and
of
episodic secretion of this ovarian hormone and that of 13,14dihydro-
15ketoPGF2a,
a
stable
metabolite
of PGF2a (Flint and Sheldrick,
1982).
Results of these studies implicate PGF2a as a major regulator of
ovarian oxytocin secretion.
secretion
However,
that
it has been demonstrated
of neurohypophyseal oxytocin is stimulated by PGE2 (Negro
Vilar et al., 1985),
which is synthesized by the uterus and is known
to regulate the life span of the corpus luteum (Silvia et al., 1984).
In
addition,
changes
plasma
in
concentrations have been found
oxytocin
concomitantly
occur
to
progesterone
and
during
estrous cycle in cattle and sheep (Sheldrick and Flint, 1981;
et
al.,
Walters
1982;
progesterone
synthesis
et
by
al.,
the
1984).
if
any,
in
altering
LH
Schams
regulates
bovine corpus luteum it is possible
that it may also affect oxytocin secretion.
LH,
Because
the
secretion
The roles
oxytocin
of
of
have
PGE2
not
and
been
investigated.
This study was conducted to determine oxytocin concentrations in
bovine corpora lutea at specific stages of the estrous cycle
examine
the
in
vitro
effects
of
PGF a
2
PGE
'
2
and
to
and LH on oxytocin
release from this tissue at the same stages of the cycle.
45
MATERIALS AND METHODS
Experiment 1
In vitro effects of PGF
the
bovine
corpus
luteum
were investigated.
years
vasectomized
PGE
2
and LH on oxytocin release from
at different stages of the estrous cycle
Sixteen Hereford and Hereford x Angus heifers
350-400
old;
2 p"
kg)
bulls.
(2
were observed twice daily for estrus using
After
exhibiting
at
least
two
consecutive
estrous cycles of normal duration (18-23 days), heifers were assigned
randomly in equal numbers to be slaughtered on each of days 4, 8,
and
of
16
12,
the estrous cycle (day of detected estrus = day 0 of the
cycle).
Approximately 20 min after
corpus
luteum
was
containing 24
mM
streptomycin,
2.5
insulin,
1984)
collected,
Hepes
slaughter,
the
placed
cold
buffer,
ug/ml
100
fungizone
U/ml
and
penicillin,
(500-600
mg
wet
luteal
original
small
divided
into
of
blotted on a dampened
200-300
Condon,
portion
and stored at 20°C
mg aliquots.
The
oxytocin.
tissue was sliced (0.3 mm thickness),
times with 40 ml of Ham's F-12,
subsequently
concentration
ug/ml
The corpus luteum
weighed and a
weight) of tissue was removed
for determination of the
remaining
100
with 5 ug/ml
supplemented
and transported to the laboratory (10 min).
the
F-12 medium
Ham's
5 ug/ml transferrin and 5 ng/ml selenium (Pate and
was dissected from the ovarian stroma,
and
in
bearing
ovary
washed four
filter
paper
These tissue
aliquots were placed into one of four incubation flasks (prepared
duplicate)
containing
in
2.955 ml of Ham's F-12 to which was added the
46
1) vehicle (control, 30 ul ethanol, 15 ul normal saline),
following:
2)
30
ng PGF2a,
3) 30 ng PGE2 and 4) 15 ng LH.
Prostaglandins F2a
and E2 were dissolved in 30 ul of ethanol and LH was dissolved in
ul
of physiological saline.
Flasks to which prostaglandins had been
added also received 15 ul physiological saline and those to which
had
CO
incubator
at
2
stoppered
2'
38°C
incubated
and
LH
All flasks were flushed with
been added received 30 ul ethanol.
95% 0 -5%
15
in
a
metabolic
Dubnoff
Incubation was terminated by immersing
for 2 h.
the flasks in ice water (4°C).
The contents were then transferred to
glass tubes and centrifuged at 3000
x
after
g,
which
the
tissue
slices
were separated from the supernatant and both were immediately
frozen
and stored at 20°C pending extraction and quantification
oxytocin.
Tissue
from
heifers
on
day
4
of
was sufficient only for
determination of initial oxytocin levels.
Experiment 2
The doseresponse effect of PGF2a on in vitro oxytocin synthesis
and(or) release from the bovine corpus luteum on day 8 of the estrous
cycle was investigated.
Six heifers similar
to
those
utilized
experiment 1 were slaughtered on day 8 of the estrous cycle.
lutea
were
collected,
and
a
Corpora
small sagittal portion of tissue was
removed and stored at 20°C for the
oxytocin concentration
in
determination
of
the
original
The remaining tissue was sliced, washed and
47
aliquots of 200-210 mg were placed into five sets of duplicate flasks
containing
2.97
ml of Ham's F-12 medium.
were as follows:
tissue,
flasks 1 and 2) unincubated and
respectively;
20 and 40 ng
PGF2a /ml
of PGF2a (30,
quantity
Flasks and the treatments
incubated
flasks 3, 4, and 5) tissue incubated with 10,
of
incubation
medium,
of
Total
respectively.
60 or 120 ng) to be added to the appropriate
flasks was dissolved in 30 ul of absolute ethanol and
volume
control
an
equivalent
this vehicle was added to each control flask.
Incubation
of tissue was performed for 2 h as described for experiment 1.
Oxytocin Extraction
Oxytocin in the incubation media was
extraction.
Tissue
oxytocin
after
using
addition
England Nuclear,
a
of
without
was extracted by a modification of the
method described by Flint and Sheldrick (1983).
homogenized
directly
assayed
Tissue samples
were
glass pestle and tube in 10 ml 1% acetic acid,
approximately
4,000
cpm
3
(U- H)oxytocin
(New
Boston, MA) to allow for calculation of recoveries.
Homogenates were centrifuged at 10,000 x g for 30 min at 4 °C and
supernatants freezedried.
the
Residues were reconstituted in 5 ml assay
buffer (0.05 M phosphate buffer, 50 mM EDTA, and 0.5 g/liter gelatin,
pH
7.5)
and centrifuged at 10,000 x g for 30 min at 4°C after which
supernatants were assayed for oxytocin.
85.0 + 5.5% (N = 12 assays).
Mean extraction recovery was
48
Oxytocin Radioimmunoassay
Oxytocin
measured
was
radioimmunoassay
by
according
to
modification of the validated method described by Schams (1983).
assay
was performed in silicone coated glass tubes (12x75mm),
0.05 M phosphate buffer with 50 mM EDTA and 0.5 g/liter
7.5,
diluent.
as
hundred
Two
a
The
using
gelatin,
pH
of extract or oxytocin
microliters
standard (450 units/mg; Calbiochem, San Diego, CA) were incubated for
24 h at 4-6°C with 100 ul antiserum (generously
donated
by
Dr.
D.
Schams, Physiological Institute, Technical University of Munich, West
Germany)
in
dilution
final
a
of
(125I)iodooxytocin (New England
buffer
were
added
continue for another 48 h.
One
serum
added,
albumin
were
then
MA)
Boston,
100
in
hundred
microliters
of
3%
bovine
followed by 400 ul dextrancoated
charcoal solution (0.66% w/v neutral norit and 0.066% w/v dextran
70
in
buffer).
centrifuged
The
tubes
ul
tube and the incubation was allowed to
each
to
Nuclear,
5,000 cpm of
About
1:100,000.
were
then
incubated
for
for
min.
T-
min and
20
at
4°C
at
3,000
centrifugation,
0.5
ml
of the supernatant was removed and counted.
x
g
15
Specificity of oxytocin antiserum used in this study
validated
(Schams
et al.,
1979;
Schams,
1983).
was
plasma
extract
interassay
to
pooled
as validated in our laboratory was 98.2 + 6.2%.
Sensitivity of the assay was 0.25 pg/tube (P<0.05,
and
previously
Mean recovery of
various amounts of standard oxytocin (0.25-24.0 pg) added
cow
Following
coefficients
of
variation
were
N
=
3.6
32).
Intra
and
9.6%,
49
respectively.
Statistical Analyses
Statistical analyses were performed according
Cochran (1980).
way
analyses
variance.
of
by
Snedecor
and
Data from experiment 1 were analyzed by one and two
oneway analysis
assessed
to
variance
of
orthogonal
Data from experiment 2 were analyzed by
differences
and
contrasts.
Linearity
among
were
groups
of the doseresponse
curve was tested for significance by computing the F values
for
the
linear and quadratic components of the sums of squares for dose.
RESULTS
Experiment 1
Luteal
concentration of oxytocin varied markedly throughout the
estrous cycle (P<0.05,
Table 1 ) increasing from
day
to
4
8
and
thereafter declining through day 12 to lowest levels on day 16.
In
vitro
release of oxytocin from luteal tissue differed among
stages of the cycle studied (P<0.01).
In parallel with
concentration of oxytocin present in the tissue,
the
in vitro release of
this hormone in response to incubation alone was greatest
compared with day 12 and lowest on day 16 (Fig. 1).
and
LH
did
not
differ
in
their
initial
on
day
8
Prostaglandin E2
ability to stimulate release of
oxytocin at each stage of the cycle nor did the quantity of
oxytocin
released
in their
in
their
presence
differ
from
that
released
50
+
TABLE 1. Oxytocin concentrations (mean
bovine
SE)
luteal tissue at different stages
of the estrous cycle
Day of estrous
No. of
Oxytocin
1
1
cycle
in
animals
(ng.g
.CL
)
a
4
4
8
4
12
4
16
4
414 +
84b
2019 + 330
589 + 101d
81±
5
a, b, c, d
Means with a different superscript letter
differ (P<0.05).
51
absence.
PGF2a, increased (P<0.05)
However, relative to the control,
the
release
16.
Luteal tissue on day 8 of the cycle not
quantities
of oxytocin on day 8 of the cycle but not on days 12 or
greater
released
of oxytocin in response to PGF2a but after incubation was
also found to contain more
(P<0.05;
only
Fig.
observation
2).
based
subsequent
It
total
consequently
that
than
conceivable
is
the
on
experiment
Nevertheless,
oxytocin
limited
that
sample
was
this
chance
a
because
size
tissue
control
of
in
the
a similar response to PGF2a was not detected.
oxytocin
levels
(tissue
medium)
+
were
greater in flasks to which PGF2a was added than that of
control flasks (P<0.05; Fig. 2).
12 and 16 was not detected,
A similar response to PGF2a on days
with total oxytocin being comparable
to
that of respective controls.
Experiment 2
Response
of luteal tissue to increasing concentrations of PGF2a
is depicted in Table 2.
of
increasing
Incubation of luteal tissue in the
concentrations
increase (P<0.05) in the
medium.
component
By
of
significant.
analysis
the
of
sums
of
quantity
PGF2a caused a significant linear
of
variance
of
Surprisingly,
presence
oxytocin
into
released
the
the linear but not the quadratic
squares
oxytocin
for
dose
found
was
concentration
to
be
(ng/g)
of
unincubated sliced tissue (2692 + 159) was significantly greater than
that of nonsliced tissue (1658 + 42) suggesting that
some
synthesis
52
"a 2500
°I. 2000
O
1500
C
12
DAY OF ESTROUS CYCLE
Fig.
1.
Oxytocin
released
(mean
+
SE)
into
incubation of luteal tissue with LH,
medium
PGF a or PGE
2
8, 12 and 16 of the estrous cycle.
after
on days
2
Responses to treatments
differed among stages of the estrous cycle (P<0.01).
Different from control (P<0.05).
2
h
53
ElCONTROL (INCUBATED TISSUE)
10 rig PGF2a / m1 (INCUBATED TISSUE )
4500
CONTROL (INCUBATED TISSUE MEDIUM)
rr.c
N
10 nq PGF2C / ml (INCUBATED TISSUE
MEDIUM
1'7W
3500
°' 2500
0
0
1500
C
500
16
12
DAY OF ESTROUS CYCLE
Fig.
2.
Concentrations of oxytocin (mean +
slices
and
in
tissue
SE)
in
luteal
tissue
+ medium after 2 h incubation with
PGF a on days 8. 12 and 16 of the estrous cycle.
2
Different from respective controls (P<0.05).
TABLE 2.
Oxytocin synthesis and(or) release by luteal tissue in response
to various levels of PGF a in vitro
2
-1
Mean oxytocin concentration (ng.g
-1
.2h
)
Treatment
Incubation medium
Tissue
a
a
Control
1824
10 ng PGF a/ml
2116
1720
b
2
20 ng PGF a/ml
a
3498
a
1889
c
b
3943
a
2332
2
40 ng PGF a/ml
Tissue + medium
1906
c
b
4163
a
b
2436
1952
4312
66
157
150
2
Common SE
Luteal tissue obtained from six heifers on day 8 of the estrous cycle was
incubated with various levels of PGF a for 2 h at 38 C.
2
a, b,c
Means within a column with a different superscript letter differ (P<0.05).
55
and(or)
processing
of
preparation for incubation.
after
incubation
did
occurred during handling in
prohormone
the
Oxytocin concentration in luteal
differ
not
treatments
among
concentration of oxytocin (tissue + medium)
total
but
different
was
tissue
(P<0.05;
Table 2).
DISCUSSION
this
In
study luteal oxytocin levels increased from day 4 to 8
of the estrous cycle and then declined through the remainder
These
cycle.
1983;
data
are
Wathes et al.,
oxytocin
in
bovine
and 10 of the cycle.
day
8
of
in
of
the
agreement with those of others (Schams,
1984) who found the highest
concentration
of
systemic blood and luteal tissue between days 5
detected
Maximal levels of luteal oxytocin
the cycle probably represent,
for the most part,
on
stored
oxytocin because greatest luteal mRNA levels for this nonapeptide are
attained on day 3
and
decline
thereafter
et
(Ivell
1985).
al.,
Results of the present study, however, differ from those of Schams et
al.
(1985a)
who reported considerably lower oxytocin concentrations
in bovine luteal tissue throughout the estrous cycle.
the corpus luteum was immediately frozen in liquid
from the animal.
In contrast,
In their study
N2
upon
removal
due to the nature of our study,
some
synthesis and(or) posttranslational processing of hormone apparently
occurred (see below) before the tissue was frozen.
Incubation of luteal tissue alone resulted
in
the
release
of
56
oxytocin
quantities
in
present in the tissue.
tissue
was
greatest
that
reflected the concentration initially
release
In vitro
on
day
oxytocin
of
Even before incubation,
increased
more
This
apparently
does
because incorporation of (
oxytocin
of
35
than
the process of slicing and washing
luteal tissue (30-40 min) increased oxytocin concentration
40%.
luteal
After 2 h incubation
8 of the cycle.
total oxytocin concentration (tissue + medium)
twofold.
from
by
about
not represent active oxytocin synthesis
ovine
S)cysteine into bovine and
luteal
and putative oxytocinprecursor was demonstrated after 12 h
incubation
only
(Swann
et
al.,
As
1984).
occurs
in
the
hypothalamus, the synthesis of luteal oxytocin involves the formation
of
an
approximately 14K precursor protein,
which is subsequently
cleaved to form neurophysin and oxytocin (Swann et
al.,
1984).
The
observed increase in oxytocin concentration may therefore represent a
posttranslational processing of the prohormone.
Prostaglandin Fe stimulated
the in vitro release of oxytocin
from luteal tissue on day 8 but not on days 12 and 16 of
In
vitro
release
of
PGF
2a
cycle.
of oxytocin from day 8 luteal tissue increased in
response to increasing levels of
Failure
the
PGF2a
in
the
incubation
medium.
to stimulate additional secretion of oxytocin from
day 12 luteal tissue in vitro is not entirely consistent with in vivo
luteal response to exogenous
Walters et al., 1983).
oxytocin
secreted
from
hormone
(Flint
and
Sheldrick,
1982;
Similarly, the relatively large quantities of
the
ovary around midcycle (Walters el al.,
57
1984;
Schams et al.,
1985b),
presumably induced by an increase
uterine PGF2a secretion (McCracken,
in
1984), might have contributed to
the low levels of this "neuropeptide" present in luteal tissue on day
12 of the cycle in our study.
If endogenous PGFa is responsible for
reducing the luteal concentration of oxytocin,
luteal
PGF
PGF2a
receptors
Addition of low levels of
to be saturated.
(lOng/m1) to incubation media, as utilized in this study, would
therefore
ineffective
be
oxytocin over control.
the
in
inducing
a
significant
release
of
Neither PGE2 nor LH was effective in inducing
release of oxytocin from luteal tissue at any stage of the cycle
studied.
The mechanisms by which oxytocin is released from the ovary
and the pituitary are apparently different.
on
PGE
one would expect most
Contrary to
its
effect
release of neurohypophyseal oxytocin (NegroVilar et al.,
1985),
2
did not stimulate the release of ovarian oxytocin.
It has been postulated that oxytocin stimulates the
PGF2a
from
the uterus (Roberts et al.,
release
of
1976) which in turn induces
the secretion of ovarian oxytocin (Flint and Sheldrick, 1982) so that
both hormones undergo a positive feedback action leading to
luteolysis.
However,
this
complete
postulated scenario does not fit all the
observations because results of this study and others (Schams,
Wathes
blood
et al.,
decline
regression
cycle.
1983;
1984) indicate that oxytocin levels in the ovary and
before
luteal
regression
begins.
In
fact
luteal
occurs when oxytocin levels are lowest during the estrous
58
It is concluded that bovine luteal tissue
oxytocin
PGF2.
of
that
is
available
release in response to
Based upon the results of the present study as well
others
cycle
as
data
it appears that synthesis is most active during the first
onehalf of the cycle.
the
immediate
for
synthesizes
actively
it
development
is
of
steroidogenesis.
Because luteal oxytocin is elevated early
possible
the
that
corpus
Oxytocin
at
this
hormone
low
plays some role in
regulation
and(or)
luteum
in
concentrations
enhanced
of
basal
steroidogenesis in cultured luteal cells obtained from pregnant
cows
but at higher concentrations it inhibited progesterone production and
the
response
Similarly,
progesterone
of
luteal cells to hCG (Tan et al.,
these
oxytocin
secretion
has
been
shown
to
inhibit
1982a,b).
LHstimulated
from small ovine luteal cells leading to the
postulate that it may serve as an
intraluteal
large and small cells (Niswender et al.,
1985).
communicator
between
A role for oxytocin
in regulating luteal steroidogenesis is supported by recent
findings
that oxytocin is also synthesized in other steroidogenic tissues such
as the testes and the adrenal cortex (Nicholson et al., 1984;
et al., 1985a).
Schams
59
EXPERIMENTS
3 AND 4: CYCLOHEXIMIDE, COLCHICINE AND CYTOCHALASIN B
DO NOT AFFECT BOVINE LUTEAL OXYTOCIN
SYNTHESIS AND RELEASE IN VITRO
INTRODUCTION
The neuropeptide
oxytocin
hormone,
is
synthesized
luteal cells of the cow and ewe (Rodgers et al., 1983;
large
by
Kruip et al.,
1985; Fields and Fields, 1986) and stored in electron dense secretory
granules (Rice and Thorburn, 1985; Guldenaar et al., 1985; Fields and
Fields,
Rao,
In vitro exocytosis of these granules
1986).
1986)
secretion
and
(Experiments 1 and 2) from
oxytocin
of
bovine luteal slices is induced by PGF2a.
that
cytoplasmic
microtubules
cytoskeleton system,
secretion
It is
generally
and microfilaments,
accepted
elements of the
are involved in the process of peptide
interacting
by
and
(Chegini
hormone
with the secretory granules to facilitate
their transport to the plasma membrane and subsequently their release
(Lacy et al., 1968;
Williams and Wolf,
1970;
Kraicer and Milligan,
1971; Olmsted and Borisy 1973; Lacy 1975).
A study of the kinetics of oxytocin
synthesis
and
by
release
bovine luteal tissue showed that highest oxytocin concentrations were
detected
day
on
8
of
estrous
the
cycle
increase in oxytocin levels after 2 h of
and
2).
throughout
However,
the
transcription
measurement
estrous
was
cycle
maximal
of
of
incubation
accompanying
(Experiments
oxytocinspecific
luteal
the
and revealed a twofold
cow
that
showed
ovulation
and
1
mRNA
gene
decreased
60
thereafter.
declined sharply around day 7,
3,
day
Highest concentrations of mRNA were detected around
reached basal levels by day 11 of
the cycle and thereafter were only barely detectable (Ivell
et
al.,
1985).
inhibition
The present study was conducted to determine whether
of
de
novo
protein
synthesis and the cellular cytoskeletal system
would inhibit this twofold increase in oxytocin levels as well as the
PGF
stimulatory effect on oxytocin release.
2a
MATERIALS AND METHODS
Experiment 3
Effects of cycloheximide and PGF20,
release
vitro
in
investigated.
vasectomized
estrous
the
day
bovine
8
Six Hereford and Hereford x
350-400
old;
from
kg)
bulls.
cycles
of
were
observed
After
normal
duration
corpus
heifers
daily
for
at
(18-23
least
were
(2
years
estrus
using
consecutive
two
days),
and
luteum
Angus
twice
exhibiting
synthesis
oxytocin
on
were
heifers
slaughtered on day 8 of the estrous cycle (day of detected
estrus
=
day 0 of the cycle).
Approximately 20 min after
corpus
luteum was collected.
slaughter,
the
the
bearing
The corpus luteum was placed into cold
Eagle's deficient Minimum Essential Medium (MEM),
Hepes buffer,
ovary
containing
24
mM
100 U/ml penicillin, 100 ug/ml streptomycin, 2.5 ug/ml
fungizone, 5 ug/ml insulin, 5 ug/ml transferrin and 5 ng/ml selenium.
In addition the medium was supplemented with MEM nonessential
amino
61
acids
(Sigma).
The
corpus
laboratory (10 min),
sliced
mm
(0.3
same medium.
was
then
dissected from
the
ovarian
thickness)
to
the
weighed,
stroma,
and washed four times with 40 ml of the
Slices were then blotted on a dampened filter paper and
subsequently divided into 200-250 mg aliquots.
placed
transported
luteum
into
one
containing 2 ml of
of
Tissue aliquots
were
five incubation flasks (prepared in duplicate)
incubation
medium
previously
(MEM,
described,
supplemented with 0.5 mM lysine, 0.1 mM methionine and 1.0 uCi/m1 [U14
C] Lleucine/m1 (308 mCi/mmol,
was added
the
following
New England Nuclear,
treatments:
1,2)
MA), to which
unincubated
none,
and
incubated controls; 3) cycloheximide (0.355 mM); 4) prostaglandin F2a
(0.042 uM) and 5) cycloheximide + prostaglandin F. All flasks were
flushed
with
95%
02-5% CO2 and stoppered.
Contents of unincubated
control flasks were then transferred to glass tubes
at
3000
x g,
after which the tissue slices were separated from the
supernatant and both were immediately frozen
Remaining
flasks
38°C for 2 h.
ice
water
centrifuged
and
and
stored
20°C.
were incubated in a Dubnoff metabolic incubator at
Incubation was terminated by immersing the
(4°C)
at
after
which
they
were
processed
flasks
in
as previously
described for unincubated controls.
Experiment 4
Effects of cytochalasin B,
oxytocin
synthesis
colchicine
and
PGF2a
on
in
vitro
and(or) release from the bovine corpus luteum on
day 8 of the estrous cycle was investigated.
Five heifers similar to
62
those utilized in experiment 3 were
estrous cycle.
Corpora
lutea
slaughtered
were
day
on
the
washed
sliced,
collected,
of
8
and
aliquots of 200-210 mg were placed into six pairs of flasks containing
2
ml
of
treatments
controls,
(0.02 mM);
Ham's
were
medium
F-12
imposed:
respectively;
(Experiments
1,2)
none,
unincubated
incubated
and
4) cytochalasin B
colchicine (0.05 mM);
3)
The following
and 2).
1
Incubation
5) PGF2a (0.042 uM) and 6) colchicine + PGF2a.
of tissue was performed for 2 h as described in experiment 3.
Total Incorporation of [
14
C]leucine into protein
Total incorporation of [14C]leucine into protein was measured by
a
modification
of the method described by Maurer and Gorski (1977).
Tissue was homogenized in 2 ml of 1% acetic acid.
vortexed,
and a 1 ml aliquot was removed for determination of
incorporation
[14C]leucine into protein.
of
used for extraction of oxytocin from tissue.
mg/ml
The homogenate was
solution
of
aliquot followed by
trichloroacetic
acid
bovine
4
ml
serum
of
(TCA).
distilled
The remaining 1 ml was
One
was
albumen
total
water
milliliter
of
10
added to the former
and
5
The mixture was vortexed,
for 5 min and then centrifuged at 800 x g for 10 min.
The
ml
of
10%
left on ice
resulting
pellet was washed twice by suspension in 2 ml of 0.2 M NaOH, addition
of
2
ml
of
water and 5 ml of TCA followed by centrifugation.
final pellet was dissolved in 2 ml of tissue
for liquid scintillation counting.
solubilizer
The
(Amersham)
63
Oxytocin Extraction and Radioimmunoassay
Oxytocin
was
from the 1 ml of 1% acetic acid tissue
extracted
homogenate as described above.
3000
x
for
g
10
was
supernatant
the
min,
reconstituted in buffer as described (Experiments 1
in the incubation medium was
Oxytocin
both
in
radioimmunoassay
tissue
assayed
(Experiments
directly
1
and
2).
freezedried
at
and
Oxytocin
and 2).
extraction.
without
was
measured by
Intraand
interassay
medium
incubation
and
centrifuged
was
homogenate
The
coefficients of variation were 3.6 and 9.6%, respectively.
Statistical Analyses
Statistical
Cochran (1980).
analyses
were
performed according to Snedecor and
Data from experiments 3 and 4 were analyzed by two
and oneway analyses of variance, respectively.
RESULTS AND DISCUSSION
incorporation
Cycloheximide inhibited (P<0.01) the
leucine
into
newly
synthesized protein by more than 90% (Table 3).
This was consistent with the well documented effect of
on
protein synthesis (Wang and Greenwald 1985;
Matsui et al., 1986).
its
retention
However,
cycloheximide
Gokal et al.,
1986;
oxytocin release into the medium and
in tissue slices after incubation was not affected by
cycloheximide (Table 4).
production
labeled
of
(tissue
At the end of
incubation,
total
oxytocin
+ medium) was approximately twofold greater than
that of unincubated control tissue levels.
This increase in oxytocin
64
14
Table 3.
Incorporation of [
C]leucine (mean + SE) in
bovine luteal tissue in vitro
Treatment
dpm/g tissue
a
740560 + 213470
Control
Cycloheximide
63200 +
10890
a
PGF a
752290 + 165530
2
Cycloheximide + PGF,
59830 +
11510
2-
Luteal tissue obtained from six heifers on day 8 of the
estrous cycle was incubated with cycloheximide (0.355 mM),
PGF,,
o-
(0.042 um) and cycloheximide + PG F
for 2 h at
38 C.
a,b
Means within a column with a different superscript letter
differ (P<0.05).
65
concentration
synthesis,
apparently
does
with
incorporation of
consistent
35
(
14
with
14
radioactivity
C
These data
C leucine into oxytocin occurred.
those
Swann
of
et
Synthesis
of
both
bovine
therefore
may
absence
of
incorporation
of
protein synthesis was inhibited.
the
fact
that
of
14
(
an
cleaved
Swann et
1984;
represent
translational processing of this prohormone because
the
precursor.
The observed increase
Ivell, 1986).
concentration
oxytocin
S)cysteine
which is subsequently
to form neurophysin and oxytocin (Ivell and Richter,
Ivell et al., 1985;
luteal
formation
the
involves
approximately 14K precursor protein,
al., 1984;
35
(
oxytocin
putative
and
oxytocin
luteal
after 12 h
However,
oxytocin
are
(1984) who reported that
al.
S)cysteine was not incorporated in either ovine or
was recovered in
was
which suggests that no
oxytocin from tissue slices,
oxytocin after 2 h of incubation.
in
This
because it could not be inhibited by cycloheximide.
is further substantiated by the fact that no
extracted
protein
novo
de
represent
not
C)leucine
This premise fits
post
occurred
it
and
a
in
while de novo
very
with
well
luteal mRNA for oxytocin is very low on day 8 of the
bovine estrous cycle (Ivell et al., 1985)
Prostaglandin Fla
oxytocin
over
provoked
control,
an
a
effect
significant
which
cycloheximide or colchicine (Tables 3,4).
was
release
not
of
luteal
inhibited
This is in agreement
by
with
previous reports that PGF2a stimulates oxytocin release in vitro from
bovine
corpora
lutea
Chegini et al., 1986).
on
day
8 of the cycle (Experiments 1 and 2;
However, this PGF2a effect was not consistent
Table 4.
Oxytocin synthesis and(or) release by luteal tissue in response to
cycloheximide and PGF a in vitro
2
1
1
Mean oxytocin concentration (ngg 2h
)
Treatment
Incubation medium
Tissue
--
1806
Unincubated control
Tissue + medium
a
Incubated control
1245
1860
2936
1782
2894
1767
2956
1428
1704
2996
32
56
75
a
Cycloheximide
1289
b
PGF a
1340
b
2
Cycloheximide + PGF a
2
Common SE
Luteal tissue obtained from six heifers on day 8 of the estrous cycle was
incubated with cycloheximide (0.355 mM), prostaglandin F
(0.042 um) and
2'
cycloheximide + prostaglandin F
for 2 h at 38 C.
2'
a, b
Means within a column with a different superscript letter differ (P<0.05).
Table 5.
Oxytocin synthesis and(or) release by luteal tissue in response
to colchicine, cytochalasin B and PGFit in vitro
1
1
Mean oxytocin concentration (ngg 2h
)
Treatment
Incubation medium
Unincubated control
Tissue
Tissue + medium
1141
a
Incubated Control
1089
1040
2095
1106
2163
1060
2274
1045
2201
1119
2309
142
199
a
1093
Colchicine
a
Cytochalasin B
1087
b
PGF a
1234
b
2
Colchicine + PGF a
1178
2
Common SE
55
Luteal tissue obtained from five heifers on day 8 of the estrous cycle
was incubated with colchicine (0.05 mM), cytochalasin B (0.02 mM), PGF a
2
(0.042 uM) and 6) colchicine + PGF a for 2 h at 38 C.
2
a, b
Means within a column with a different superscript letter differ (P<0.05).
68
with the results of Hirst et al.
effect
oxytocin
on
(1986) who found that PGF2a had
from
release
ovine
tissue
luteal
in vitro.
Prostaglandin Fla also failed to induce oxytocin release from
luteal
no
bovine
slices on days 12 and 16 of the estrous cycle (Experiment 1).
Prostaglandin Fla may
therefore
not
universally
be
effective
in
stimulating the release of oxytocin from luteal tissue.
Neither
cholchicine
nor
cytochalasin
release into the medium or
its
retention
incubation
(Table
through direct binding to tubulin dimers (Borisy
Wilson
et
Margolis
1974;
al.,
and
1971) and insulin (Lacy et
oxytocin
duration
of
equilibrium
be
al.,
thyroxine
1968),
with
Failure
1968).
of
elicit
At
a
concentrations,
low
an
important
(Khar et al., 1979;
et
al.,
components
colchicine
In
of
cytoskeleton,
microfilament
hormone
Adams and Nett, 1979;
1985).
therefore
may
are
in the secretion of hormones.
role
a drug that inhibits
gonadotropinreleasing
blocked
to
effect on oxytocin release.
colchicine
actincontaining
play
Cytochalasin B,
colchicine
tubulin is not reached until 6-8 h (Wilson et al.,
to
Microfilaments,
to
(Williams
release in the present study may be due to the short
incubation.
necessary
Lewis
1977) and blocks the
Preincubation or incubation for a longer time
thought
1967;
Tayler,
1970), adrenocorticotropic hormone (Kraicer and Milligan,
and Wolff,
1974).
and
Wilson,
release of histamine (Gillespie et al.,
block
after
slices
reportedly disrupts microtubules
Colchicine
5).
tissue
in
oxytocin
inhibited
B
contrast,
polymerization,
(GnRH)induced
LH
release
Pickering and Fink, 1979;
Liu
and
Jackson,
(1986)
69
demonstrated that cytochalasin B did not effect either basal or GnRH
stimulated LH release but inhibited GnRHstimulated LH glycosylation.
In the present study cytochalasin B had no effect on either basal
or
PGF astimulated oxytocin release.
2
It
is
concluded
that short term release of luteal oxytocin in
vitro is neither contingent upon de novo protein synthesis nor can it
be
interrupted
integrity.
Over
by
exposure
to
a period of 2 h,
drugs
that
inhibit
cytoskeletal
oxytocin synthesis and release by
bovine luteal tissue on day 8 of the estrous cycle may depend
on the posttranslational processing of oxytocin prohormone.
mainly
70
GENERAL DISCUSSION
bovine
attention.
the
corpus luteum have recently received considerable
ovine
and
from
release
Factors that regulate oxytocin synthesis and
In this section an attempt will be made
discuss
to
the
results of the four experiments conducted in the present research and
compare
with
them
results
of
research
from
other laboratories.
Results of Experiment 1 of the present study demonstrated that luteal
highest
oxytocin concentrations in the cow were
estrous
cycle.
on
day
8
greatest
the
These data are consistent with the results of Wathes
that
(1984) and Sheldrick and Flint (1983) who reported
et al.
of
concentrations
of
luteal
the
were found during the
oxytocin
early luteal phase of the cycle in the cow and ewe, respectively.
In Experiment 1, PGF2ainduced a significant in vitro release of
oxytocin from bovine luteal tissue collected on day 8 of the
cycle.
Similarly,
Chegini
and
estrous
Rao (1986) found that PGF2ainduced
migration and exocytosis of electrondense
secretory
granules
that
contained oxytocin (Rice and Thorburn, 1985; Fields and Fields, 1986)
from
bovine large luteal cells in vitro.
The results of the present
study also agree with those of Barrett et al.
doses
of
PGF a
2
consistent
found
low
to cause a slight increase in oxytocin release from
bovine luteal cells cultured for 24 h.
not
(1987) who
However,
with those of Hirst et al.
these
results
are
(1986) who reported that
PGF a had no effect on oxytocin release from ovine luteal
slices
on
2
days 8-10 of the estrous cycle.
In addition,
PGF2a failed to induce
71
a significant release of oxytocin in vitro from bovine corpora
on
days
and
12
of
16
The effect of
cycle in Experiment 1.
the
lutea
prostaglandin F2a on oxytocin release from luteal cells may therefore
vary with the stage of the estrous cycle.
Luteinizing hormone and PGE2 had no effect on
These results agree with those of
at any stage of the cycle studied.
Chegini
Rao
and
(1986) who could find no
and Hirst et al.
(1986)
effect of these hormones on oxytocin release from
tissue
luteal
in vitro.
However,
results of Barrett et al.
stimulating
oxytocin
bovine
oxytocin
(1987) who reported that LH was capable of
release
bovine luteal cells in culture.
from
release
vitro.
in
were
incubated
for
2
Hirst
elicit LH
et
al.
and in the
1
luteal
(1986)
which may not have been of
only,
h
Experiment
In
studies of Chegini and Rao (1986) and
slices
ovine
and
they are not consistent with the
This suggests that a longer time (24 h) may be needed to
induced
release
oxytocin
sufficient duration to demonstrate the effect of LH.
Oxytocin
demonstrated
synthesis
in
by
bovine
Experiments
confirmed by Barrett et al.
1,2,3
(1987).
luteal
slices
and
These
4.
However,
was
data
clearly
have been
increases in oxytocin
concentrations by bovine luteal slices on day 8 of the cycle does not
appear
to
represent
de
novo
synthesis because it occurred in the
absence of incorporation of labeled leucine and during the inhibition
of de
novo
oxytocin
protein
synthesis.
Ivell
et
al.
(1985)
found
that
mRNA concentrations in bovine luteal tissue were highest on
day 3 of the cycle, very low on day 7 and reached basal levels by day
72
11.
This suggests that the major portion of oxytocin in
corpus
the
bovine
luteum is synthesized around day 3 of the cycle and stored in
the form of oxytocin prohormone which
is
processed
further
during
later stages.
Further
research
should be undertaken to elucidate the factors
that regulate the expression of oxytocin gene
synthesis
and
release
from
the
as
corpus luteum.
effects of oxytocin on the corpus luteum
cycle in general need to be examined.
function
well
as
oxytocin
Furthermore,
and
the
the
estrous
73
BIBLIOGRAPHY
Interaction of GnRH with anterior
Adams, T.E. and T.M.
Nett.
1979.
microtubules and
pituitary.
Role of divalent cations,
III.
Reprod.
microfilaments in the GnRH activated gonadotroph. Biol.
21:1073-1086.
Direct inhibition of
E.Y.
and A.J.W.
1981a.
Hsueh.
testicular androgen biosynthesis revealing antigonadal activity
of neurohypophyseal hormones. Nature 293:650-652.
Adashi,
Adashi,
E.Y.
and
A.J.W.
Hsueh.
1981b.
testicular
androgen
biosynthesis
mediation through pressorselective
sites.
Endocrinology 109:1793-1795.
Direct inhibition of
arginine vasotocin:
recognition
testicular
by
Direct inhibition of
E.Y.
and A.J.W.
1982.
Hsueh.
Studies
testicular androgen biosynthesis by arginine vasotocin.
on mechanisms of action. J. Biol. Chem. 257:1301-1308.
Adashi,
Effect of
1984.
Smith.
Zahler and M.F.
prostaglandin Fix on the adenylate cyclase and phosphodiesterase
activity of ovi ne corpora lutea. J. Anim. Sci. 58:955-962.
Agudo,
L.Sp.,
W.L.
Kaltenbach and
Akbar, A.M., L.E.
C.C.
Reichert,
Dunn,
Jr.,
T.G.
G.D.
Niswender.
1974.
Serum levels of folliclestimulating
J. Anim. Sci. 39:360hormone during the bovine estrous cycle.
365.
Alila, H.W.
and W.
Hansel. 1984. Origin of different cell types in
the bovine corpus luteum as characterized by specific monoclonal
antibodies.
Biol. Reprod. 31:1015-1025.
Amoroso,
E.G.
1951.
The interaction of the trophoblast and
J.
Anat.
endometrium at the time of implantation in the sheep.
85:428-429.
Oxytocin on
1965.
Melampy.
Anderson, L.L., A.M.
Bowerman and R.M.
ovarian function in cycling and hysterectomized heifers. J.
Anim.
Sci. 24:964-968.
Localization of relaxin in the
Long.
1978.
Anderson, M.L. and J.A.
pregnant rat. Bioassay of tissue extracts and cell fractionation
Reprod. 18:110-117.
studies.
Biol.
Steinetz.
O'Byrne and B.G.
Perezgrovas,
E.M.
L.L.,
R.
and beef cattle.
1982.
Biological actions of relaxin in pigs
Ann.
N.Y.
Acad.
Sci. 380:131-150.
Anderson,
74
Armstrong, D.T. 1975.
Role of prostaglandins in follicular responses
Biophys.
Biochim.
Biol.
Anim.
to luteinizing hormones.
Ann.
15:181-190.
Armstrong,
Alteration of the bovine
1959.
D.T.
and W.
Hansel.
estrous cycle with oxytocin. J. Dairy Sci. 42:533-542.
Armstrong,
Preovulatory elevation of
D.T.
and J.
Zamecnik.
1975.
rat ovarian prostaglandins F and its blockade by indomethacin.
Mol.
Cell. Endocrinol. 2:125-131.
1981.
C.G. 6Smith and A.V. Schally.
Asch, R.H., T.M.
SilerKhodr,
Luteolytic
effect
of
DTrp luteinizing hormonereleasing
Clin.
J.
hormone in the rhesus monkey (Macaca mulatta).
Endocrinol. Metab. 52:565-571.
Aten,
Behrman 1986a.
Presence of GnRHlike ovarian hormone (GLOH) in human ovaries.
Proc. of the 33rd Mtg. Soc. Gynaecol. Invest. Toronto, Canada.
Aten,
1986b.
R.F.,
Wolin.
A.T.
Behrman and D.L.
Williams,
H.R.
protein(s):
Ovarian
hormonelike
gonadotropinreleasing
Endocrinology 118:961-967.
demonstration and characterization.
R.F.,
M.L.
Polan,
B.
Bayless
and
H.R.
D.T.
1978.
Local
uteroovarian relationships. In: D.B.
Lamming (eds.).
Foxcroft and G.E.
Crighton, N.B. Haynes, G.R.
Control of Ovulation. Butterworths, LondonBoston. pp 217-236.
Baird,
Changes in the secretion of
Baird, D.T. and R.J. Scaramuzzi.
1976.
ovarian steroids and pituitary luteinizing hormone in the peri
J.
the effect of progesterone.
ovulatory period in the ewe:
Endocrinol. 70:237-245.
DJ., I. Swanston and R.J. Scaramuzzi. 1976. Pulsatile
release of LH and secretion of ovarian steroids in sheep during
Endocrinology 98:1490the luteal
phase of the estrous cycle.
Baird,
1496.
Effect of
Wathes. 1987.
DenningKendall and D.C.
Barrett, J., P.A.
gonadotropins and PGF2a on oxytocin release by bovine ovarian
Study Fertil.
Soc.
1987.
cells in vitro.
Annual Conference,
75.
Godkin
J.D.
Lewis,
G.S.
Bartol, F.F., R.M.
Bazer,
Roberts, F.W.
Characterization of proteins produced
Thatcher. 1985.
and W.W.
Reprod.
Biol.
in vitro
by periattachment bovine conceptuses.
32:681-693.
75
Beck,
of serum
T.W.
1977.
Estradiol control
and E.M.
Convey.
luteinizing hormone concentrations in the bovine.
J. Anim. Sci.
45:1096-1101.
Behrman,
H.R.,
mechanism
S.L.
of
the
hormonereleasing
Preston,
1980.
Cellular
Hall.
of
luteinizing
action
Endocrinology
in the corpus luteum.
and
A.K.
antigonadotropic
hormone
107:656-664.
Berridge, M.J. and R.F. Irvine. 1984.
second
messenger in cellular
312:315-321,
Inositol triphosphate, a novel
transduction.
Nature
signal
Bex, F.J., A.
Resistance of the mouse
Corbin and E. France.
1982.
to the antifertility effects of LHRH agonists.
Life Sci.
30:1263-1269.
Evidence
Vale. 1985.
Birnbaumer, L., N.
Rivier and W.
Shahabi, J.
for a physiological
releasing hormone
role of gonadotropin
(GnRH)
or GnRHlike material
Endocrinology
in
the ovary.
116:1367-1370.
Bjersing,
L.,
F.
Naftolin,
M.F.
Moor,
Hay,
G.Kann,
R.M.
R.J.
Scaramuzzi,
1972.
Changes in
R.V.
Short and E.V.
Younglai.
gonadotrophins,
ovarian steroids and follicular morphology in
sheep at oestrus.
J.
Endocrinol. 52:465-479.
Bolt,
D.J.,
H.E.
Kelley and
estradiol in cycling ewes.
Release of LH by
Biol. Reprod. 4:35-40.
H.W.
Hawk.
1971.
Borisy,
G.G.
and J.
Taylor.
1967.
colchicine.
Colchicine binding
mitotic apparatus. J.
The mechanism of action of
to sea urchin eggs and the
Cell Biol. 34:535-548.
Brinkley, H.J.
and A.V.
Effect of oxytocin
Nalbandov.
1963.
ovulation in rabbits and rats. Endocrinology 73:515-517.
on
and J.J.
Reeves. 1983. Absence of specific luteinizing
hormone releasing hormone receptors in ovine, bovine and porcine
ovaries.
Biol. Reprod. 29:1179-1182.
Brown, J.L.
Brunner,
M.A.,
1969.
L.E.
Hansel.
Donaldson and W.
hormones and luteal function in hysterectomized
heifers.
J. Dairy Sci. 52:1849-1854.
Exogenous
and intact
Thimonier,
Cahill, L.P., J.
J.
Blanc,
Saumande, J.P. Ravault, M.
Hormonal and follicular
J.C.
Mariana and P.
1981.
Mauleon.
relationships in ewes of high and low ovulation rates. J.
Reprod.
Fertil. 62:141-150.
76
Carlson,
J.C.,
Effect of LH on
N.
Kazama and W. Hansel. 1971.
and
peripheral
in
intact
progesterone
concentrations
Endocrinolgy 89:1530-1533.
hysterectomized heifers.
Carson,
R.S.,
Clarke and H.G.
Burger.
1981.
J.K.
Findlay,
I.J.
Estradiol, testosterone, and androstenedione in ovine follicular
fluid during growth and atresia of ovarian follicles.
Biol.
Reprod. 24:105-113.
R.F.,
K.L.
1980.
Chorionic
Yen.
Sheehan and S.S.
gonadotropin prevents LRFagonistinduced luteolysis in the
human.
Contraception 21:471-478.
Casper,
Casper,
Induction of luteolysis in the
S.S.
1979.
Yen.
human with a longacting analog of luteinizing hormonereleasing
factor.
Science 205:408-410.
R.F.
Chakraborty,
1974.
and
P.K.,
Serum
T.E.
Adams,
and pituitary
with LHRH/FSHRH.
J.
Chegini,
N.
and Ch.V.
Rao.
granules
in
bovine
Reeves.
Tarnaysky and J.J.
G.K.
concentrations in ewes infused
LH
Anim. Sci. 39:1150-1157.
Dynamics of nuclear associated
after treatment with
cells
Reprod. 34
Biol.
prostaglandins, gonadotropins and cyclic AMP.
(Suppl. 1):207.
1986.
luteal
Chenault, J.R. 1983. Response of bovine corpora lutea to intrauterine
prostaglandin E2 infusion. J. Anim. Sci. 57(Suppl. 1):313.
Clark,
M.R.,
1980.
LeMaire.
Thibier,
J.M.
Marsh and W.J.
Stimulation
of
prostaglandin
accumulation by luteinizing
rat
hormonereleasing hormone (LHRH) and LHRH analogs in
granulosa cells in vitro.
Endocrinology 107:17-23.
C.
Clayton,
R.N.,
1979.
J.P.
Harwood and K.J. Catt.
releasing hormone analogue binds to luteal cells
progesterone production.
Nature 282:90-92.
Clayton, R.N.
and I.T.
Huhtaniemi.
releasing hormone receptors in
299:56-59.
Gonadotropin
and
inhibits
1982.
Absence of gonadotropin
human
gonadal
tissue.
Nature
Effect of prosta
1978.
Colcord, P&L., G.L.
Hoyer and C.W. Weems.
glandin E2 (PGE2) as an antiluteolysin in estrogeninduced
luteolysis in ewes. J. Anim. Sci. 47(Suppl. 1):352.
Convey, E.M.
review.
Neuroendocrine relationships in farm animals:
J. Anim. Sci. 37:745-757.
1973.
a
77
Carruthers and
T.D.
V.
Padmanabhan,
Convey,
Kesner,
E.M.,
J.S.
Luteinizing hormone releasing hormoneinduced
T.W.
Beck. 1981.
release of luteinizing hormone from pituitary explants of cows
Endocrinol.
J.
killed before or after oestradiol treatment.
88:17-25.
Homeida. 1982. Plasma concentrations of 13,14
F9a and progesterone during
dihydro-15keto
prostaglandin
oxytocininduced estrous in the goat. Theriogenology 18:453-460.
Cooke, R.G. and A.M.
Prevention of the luteolytic
1983.
Cooke,
R.G.
and A.M.
Homeida.
action of oxytocin in the goat by inhibition of prostaglandin
Theriogenology 20:363-365.
synthesis.
Suppression of prostaglandin
1985.
and A.M.
Homeida.
release and delay of luteolysis after active immunization
against oxytocin in the goat. J. Reprod. Fertil. 75:63-68.
Cooke,
R.G.
F2'
Cooke, R.G.
goat.
Knifton.
and A.
1981.
Theriogenology 16:95-97.
Oxytocininduced estrous in the
Corner, G.W. 1919. On the origin of the corpus luteum of the sow from
both granulosa and theca interna. Am. J. Anat. 26:117-183.
Luteinizing hormone release
1977.
Crighton, D.B.
and J.P.
Foster.
after two injections of synthetic luteinizing hormone releasing
hormone in the ewe.
Endocrinol. 72:59-67.
J.
Dawood,
M.Y.
and F.S.
KhanDawood. 1986. Human ovarian oxytocin:
J.
hormones. Am.
its source
and relationship to steroid
Obstet.
Gynecol. 154:756-763.
Dobson, H. 1978.
in the cow.
Plasma gonadotrophins and oestradiol during oestrus
J. Reprod. Fertil. 52:51-53.
Histological study of bovine
1965a.
Donaldson, L.E. and W. Hansel.
corpora lutea. J. Dairy Sci. 48:905-909.
Hansel. 1965b. Prolongation of the life span
Donaldson, L.E.
and W.
of the bovine corpus luteum by single injections of bovine
luteinizing hormone. J. Dairy Sci. 48:903-904.
The
luteotropic properties of luteinizing hormone and the nature of
Dairy.
Sci.
oxytocin induced luteal inhibition in cattle. J.
48:331-337.
Donaldson,
L.E.,
W.
Donaldson,
and A.
L.E.
oxytocin on corpus
Fertil. 17:373-383.
Hansel
and
L.D.
VanVleck.
1965.
The effect of exogenous
1968.
Takken.
Reprod.
luteum function in the cow. J.
78
1961.
Donovan, B.T.
The role of the uterus in the regulation of the
oestrous cycle. J. Reprod. Fertil. 2:508-510.
Dorflinger, L.J., P.J. Albert, A.T. Williams and H.R. Behrman. 1984.
Calcium
is
an
inhibitor of luteinizing hormonesensitive
adenylate cyclase in the luteal cell. Endocrinology 114:12081215.
Duncan,
G.W.,
Bowerman L.L. Anderson,
A.M.
Melampy.
1961.
Factors influencing in
progesterone.
Endocrinology 68:199-207.
W.R.
vitro
Hearn and R.M.
synthesis of
Duncan, G.W., A.M.
Bowerman, W.R. Hearn and R.M. Melampy. 1960. In
Proc.
vitro synthesis of progesterone by swine corpora lutea.
Soc.
Exp.
Biol.
Med. 104:17-19.
Eiler, H. and A.V. Nalbandov. 1977. Sex steroids in follicular fluid
of
pigs.
cycle
and blood
the
estrous
plasma
during
Endocrinology 100:331-338.
Ellinwood, W.E., T.M. Nett and G.D. Niswender. 1979a.
the
corpus
luteum
of early pregnancy in
Luteotropic properties of embryonic homogenates.
21:281-288.
Maintenance of
I.
the ewe.
Biol.
Reprod.
Maintenance of
Ellinwood, W.E., T.M. Nett and G.D. Niswender. 1979b.
II.
the
corpus
luteum of early pregnancy in the ewe.
Prostaglandin secretion by the endometrium in vitro and in vivo.
Biol.
Reprod. 21:845-856.
Enders,
A.C.
1973.
8:158-182.
Cytology
of the corpus luteum.
1971.
Erb, R.E., R.D. Randel and C.J.
Callahan.
cycle.
changes during the reproductive
(Suppi.
1):80-106.
L.L.
1978.
Vertebrate Ovary.
Espey, L.L.
sis.
Reprod.
Female sex steroid
J.
Development and senescence of
Erickson,
B.H.
1966.
bovine ovary. J. Anim. Sci. 25:800-805.
Espey,
Biol.
Anim.
the
Sci.
32
postnatal
Jones,
R.E.
(eds.).
In:
Ovulation.
Plenum Press, New York. pp. 503-532.
The
Ovulation as an inflammatory reaction a hypothe
1980.
Biol.
Reprod. 22:73-106.
Eyster, K.M., J.S. Ottobre and R.L. Stouffer. 1985. Adenylate cyclase
in the corpus luteum of the rhesus monkey. III. Changes in basal
and gonadotropinsensitive activities during the luteal phase of
the menstrual cycle. Endocrinology 117:1571-1577.
79
Passive
1976.
Fairclough,
Peterson.
R.J.,
J.F.
Smith and A.J.
on
effect
and
its
oestradiol -17B
immunization
against
Reprod.
J.
luteolysis,
oestrus and ovulation in the ewe.
Fertil. 48:169-171.
Fields, P.A. 1984. Ultrastructural localization of relaxin in corpora
lutea of pregnant,
pseudopregnant and cycling gilts using
porcine relaxin antiserum and goat antirabbit IgGcolloidal
gold. Biol. Reprod. 30(Suppl. 1):116.
Roberts and M.J.
Fields,
R.F.
P.A.,
R.K.
Eldridge,
A.R.
Fuchs,
Fields.
1983.
Human placental and bovine corpora luteal
oxytocin.
Endocrinology 112:1544-1546.
Fields,
M.J.
and
neurophysin in the
Fields.
1986.
Luteal
localization in
immunocytochemical
ewe:
secretory granules of the large luteal cell.
P.A.
nonpregnant cow
and
membranebounded
Endocrinology 118:1723-1725.
Larkin.
CastroHernandez and L.H.
Fields,
M.J.,
P.A.
Fields,
A.
1980.
Evidence for relaxin in corpora lutea of late pregnant
cows.
Endocrinology 107:869-876.
Octadecylsilica
1982.
Fields,
M.J.,
Fields.
R.
Roberts and P.A.
cellulose isolation of bovine and porcine
and carboxymethyl
relaxin.
Sci. 380:36-46.
Ann.
N.Y.
Acad.
Maule
Davis,
F.M.
A.J.
Findlay, J.K., N.
Burton,
Ackland,
R.D.
Protein,
1981.
Walker,
D.E.
Heap.
Walters
and
R.B.
and
caruncular
in
prostaglandin and
steroid
synthesis
intercaruncular endometrium of sheep before implantation. J.
Reprod. Fertil. 62:361-377.
Fitz,
Fitz,
Sawyer and G.D. Niswender.
Characterization of two steroidogenic cell types in the
corpus luteum.
Biol. Reprod. 27:703-711.
T.A.,
M.H.
Mayan,
H.R.
1982.
ovine
1984.
Niswender.
Mayan and G.D.
Interactions of prostaglandins with subpopulations of ovine
luteal
cells.
II.
Inhibitory effects of PGF2a and protection
by PGE2.
Prostaglandins 28:127-138.
T.A.,
E.J.
Mock,
M.H.
1982.
Effect of
P.W.
and G.D.
Niswender.
progesterone secretion and adenylate cyclase activity
Prostaglandins 23:803-818.
luteal tissue.
Fletcher,
and E.L.
1982.
A.P.F.
Sheldrick.
oxytocin is stimulated by prostaglandin.
Flint,
PGF2a
in
on
ovine
Ovarian secretion of
Nature 297:587-588.
80
Flint,
A.P.F.
Evidence for a systemic
and E.L.
Sheldrick.
1983.
J.
role for ovarian oxytocin in luteal regression in sheep.
Reprod.
Fertil. 67:215-225.
Recherches experimentales sur la
Foote, W.D. and C.
Thibault. 1969.
maturation in vitro des ovocytes de truie et de veau. Ann.
Biol.
Anim.
Biochim. Biophys. 9:329-349.
Frieden, E.H.,
P.
Vasilenko III and W.C. Adams.
porcine relaxin in cycling and pregnant rats.
Sci. 380:174-177.
Effects of
1982.
N.Y.
Acad.
Ann.
Garverick, H.A., M.F. Smith, R.G. Elmore, G.L. Morehouse, L. Sp. Agudo
and W.L.
Zahler.
1985.
Changes and interrelationships among
and
luteal
cyclase
activity
LH
receptors,
adenylate
phosphodiesterase activity during the bovine estrous cycle. J.
Anim. Sci. 61:216-623.
Thibault.
Szollosi and C.
D.
Menezo, P.
Rombauts,
vitro studies of oocyte maturation and follicular
Biophys.
Anim.
Biochim.
metabolism in the pig. Ann.
Biol.
19:1521-1535.
Gerard, M., Y.
1979.
In
1968. Histamine release
Gillespie, E., R.
Levine and S. Malawista.
from rat peritoneal mast cells:
inhibition by colchicine and
Ther.
Exp.
potentiation by deuterium oxide. J.
Pharmacol.
164:158-165.
Ginther,
0.J.,
Janakiraman and L.E.
C.O.
Woody,
Mahajan,
K.
S.
Casida. 1967.
Effect of oxytocin administration on the oestrous
Reprod.
J.
cycle of unilaterally hysterectomized heifers.
Fertil. 14:225-229.
1985.
Cytosolic
J.D.,
T.A.
Fitz and G.D.
Niswender.
receptor for estradiol in the corpus luteum of the ewe:
variation throughout the estrous cycle and distribution between
large and small steroidogenic cell types. Biol. Reprod. 31:967-
Glass,
974.
1973.
Pope.
Glencross,
R.G.,
I.B.
Munro,
B.E.
Senior and G.S.
Concentrations of estradiol-17g, oestrone and progesterone in
jugular venous plasma of cows during the oestrous cycle and in
early pregnancy.
Acta Endocrinol. 73:374-384.
The demonstration that PGF21 is the
luteolysin in the ewe. J. Reprod. Fertil. 38:261-271.
Goding,
J.R.
1974.
uterine
81
Godkin,
J.D.,
Roberts.
Sessions and R.M.
low
Purification and properties of a major,
molecular weight protein released by the trophoblast of sheep
blastocysts at day 13-21.
J. Reprod. Fertil. 65:141-150.
F.W.
1982.
Bazer,
J.
Moffatt,
F.
Godkin,
J.D.
and
S.
McGrew.
1986.
Characterization of
conceptus protein production during early pregnancy.
bovine
Biol.
Reprod. 34(Suppl. 1):148.
Gokal,
P.K., A.H.
Thompson.
Cavanaugh and E.A.
of cycloheximide upon transcription of rRNA,
genes.
J.
Biol. Chem. 261:2536-2541.
1986.
The effects
and tRNA
5 S RNA,
Uterine blood flow during
F.D.
and S.G.
1970.
Anderson.
early ovine pregnancy. Am. J. Obstet. Gynecol. 106:30-38.
Griess,
Guldenaar,
S.E.F.
and B.T.
1985.
Immunocytochemical
Pickering.
Cell
evidence for the presence of oxytocin in rat testis.
Tissue Res. 240:485-487.
Immuno
Guldenaar, S.E.F.,
1985.
D.C.
Pickering.
Wathes and B.T.
cytochemical
evidence
for
the presence of oxytocin and
neurophysin in the large cells of the bovine corpus luteum.
Cell Tissue Res. 237:349-352.
Hafez,
1980.
E.S.E.,
C.
Thibault.
and
M.O.
Levasseur
Folliculogenesis,
In:
E.S.E.
egg maturation and ovulation.
Hafez (ed.).
Lee and Febiger.
Reproduction in Farm Animals.
Philadelphia. pp. 150-166.
Hafs,
H.D.
and D.T.
Armstrong.
1968.
Corpus luteum growth and
progesterone synthesis during the bovine estrous cycle. J.
Anim.
Sci. 27:134-141.
Hamberger,
Sjogren.
Khan and A.
Dennefors, I.
Nilsson, B.
in
lutea
1979.
Cyclic AMP formation by isolated human corpora
Prostaglandins 17:615response to hCGinterference by PGF2a.
L., L.
621.
Hammond, J. and P.
Bhattacharya.
cow.
J.
Agr. Sci. 34:1-15.
1944.
Control of ovulation in the
Hansel, W. 1971.
Survival and gonadotrophin responsiveness of luteal
Karolinska Symp.
cells in vitro.
In:
A.
Diczfalusy (ed.).
in
Res.
vitro Methods
Methods in Reprod.
In
Endocrinol.:
Reprod.
Stockholm, Sweden [Also Suppl.
Cell Biol.
pp 295-317.
153, Acta Endocrinol. (Kbh)].
Corpora
1973.
W.,
P.W.
Lukaszewska.
Concannon and J.H.
Biol. Reprod. 8:222-245.
lutea of the large domestic animals.
Hansel,
82
Hansel, W.
and E.M.
Physiology of the estrous cycle.
Convey. 1983.
J.
Anim.
Sci. 57(Suppl 2):404-424.
Hansel,
W.
and J.P.
Dowd.
corpus luteum function.
of
Luteal inhibition in the bovine
and W.C.
Wagner.
1960.
and
result of oxytocin injection, uterine dilatation,
Hansel, W.
as
New concepts of the control
J. Reprod. Fertil. 78:755-765.
1986.
a
intrauterine infusion of seminal and preputial fluids.
Sci. 43:796-805.
J. Dairy
Harms,
1969.
Progesterone
P.G.,
G.D.
Niswender and P.V. Malven.
secretion during luteal inhibition by
and luteinizing hormone
exogenous oxytocin.
Harwood,
J.P.,
R.N.
Biol. Reprod. 1:228-233.
Clayton
and
K.J.
Catt.
1980.
Ovarian
gonadotropinreleasing hormone receptors. I. Properties and
Endocrinology 107:407-413.
inhibition of luteal cell function.
Heath,
Nixon.
Shanks and J.
E.,
R.
P.
Weinstein,
B.
Merritt,
1983.
Effects of prostaglandins on the bovine corpus luteum:
Biol.
granules,
lipid inclusions and progesterone secretion.
Reprod. 29:977-985.
R.V.
Helmer,
Anthony,
Thatcher,
F.W.
Hansen,
S.D.,
P.J.
W.W.
Bazer and R.M.
Roberts.
Identification of bovine
1987.
trophoblast protein-1,
a
secretory protein immunologically
Reprod.
Fertil.
J.
related to ovine trophobast protein-1.
79:83-91.
Henderson, K.M.
and K.P. McNatty. 1975.
explain the mechanism of luteal
9:779-797.
A biochemical hypothesis to
Prostaglandins
regression.
1977.
Henderson,
Baird.
and
D.T.
K.M.,
R.J.
Scaramuzzi
the
Simultaneous infusion of prostaglandin E2 antagonizes
Endocrinol.
luteolytic action of prostaglandin F2a in vivo. J.
72:379-383.
1982.
Raab.
F.
Asem and B.
F.,
Lintner,
E.K.
Synergistic effect of gonadotropin releasing hormone on LH
stimulated progesterone production in granulosa cells of the
domestic fowl (Gallus domesticus) Gen. Comp. Endocrinol. 48:117-
Hertelendy,
122.
Evidence for
HildPetito, S., S. Shiigi and R.L. Stouffer. 1986.
multiple cell populations in the primate corpus luteum. Biol.
Reprod. 34(Suppl 1)027.
83
1986.
Rice,
G.
Jenkin and G.D. Thorburn.
J.J.,
G.E.
Secretion of oxytocin and progesterone by ovine corpora lutea in
Proc.
Soc.
guinea pig.
vitro.
Biol.
Reprod.
35:1106-1114.
Exper. Biol. Med. 23:661-663.
Hirst,
Experimental relaxation of the pubic ligament of
F.L.
1926.
the guinea pig. Proc. Soc. Exper. Biol. Med. 23:661-663.
Hisaw,
Experimental relaxation of the symphysis pubis of
Hisaw, F.L.
1927.
the guinea pig. Anat. Rec. 37:126.
Uterine luteolytic hormone: a
Horton, E.W.
1976.
and N.L.
Boyser.
56:595physiological role for prostaglandin F2a. Physiol. Rev.
651.
Howland, B.E., A.M. Akbar and F. Stormshak.
and
luteal weight
in
ewes following
estradiol.
Biol. Reprod. 5:25-29.
Serum LH levels
single injection of
1971.
a
1978.
Effects
Weems.
G.L.,
M.L.
Colcord and C.W.
prostaglandin El on estrogeninduced luteolysis in ewes.
Anim. Sci. 47(Suppl. 1):367.
Hoyer,
of
J.
1986.
Size
P.B.,
and G.D.
Niswender.
P.L.
Keyes
distribution and hormonal responsiveness of dispersed rabbit
luteal cells during pseudopregnancy. Biol. Reprod. 34:905-910.
Hoyer,
Welsh, Jr. 1984.
Hsueh, A.J.W., E.Y. Adashi, P.B.C. Jones and T.H.
ovarian
cultured
Hormonal regulation of the differentiation of
Endocr. Rev. 5:76-127.
granulosa cells.
Extrapituitary action of
A.J.W.
1979.
and G.F.
Erickson.
gonadotropinreleasing hormone: direct inhibition of ovarian
steroidogenesis. Science 204:854-855.
Hsueh,
Effect of an antagonistic analog
Hsueh, A.J.W. and N.C.
Ling. 1979.
of gonadotropin releasing hormone upon ovarian granulosa cell
function. Life Sci. 25:1223-1229.
Direct inhibitory
1980.
Hsueh, A.J.W., C.
Wang and G.F.
Erickson.
follicle
upon
hormone
effect of
gonadotropinreleasing
stimulating hormone induction of luteinizing hormone receptor
Endocrinology
and aromatase activity in rat granulosa cells.
106:1697-1705.
Effect of PGE on normal
Huecksteadt,
1978.
and C.W.
T.P.
Weems.
1
luteolysis and PGE1 or PGE in IUDinduced luteolysis in ewes.
2
Pharamcologist 20:233-234.
84
1982.
Prostaglandin
J.H.,
Manns and W.D. Humphrey.
J.G.
production by ovine embryos and endometrium in vitro. J.
Reprod.
Fertil. 65:299-304.
Hyland,
Pexton.
1975.
Butcher and J.E.
E.K.,
W.J.
Smutny, R.L.
Effects of intrafollicular injections of prostaglandins in non
pregnant and pregnant ewes. J. Anim. Sci. 41:1098-1104.
Inskeep,
Development of antral follicles
Ireland, J.J. and J.F. Roche. 1982.
changes in
in cattle after prostaglandininduced luteolysis:
and gonadotropin
serum hormones,
steroids in follicular fluid,
receptors.
Endocrinolgy 111:2077-2086.
Development of nonovulatory
changes in steroids in follicular
antral follicles in heifers:
Endocrinology 112:150fluid and receptors for gonadotropins.
Ireland,
J.J.
and
J.F.
Roche.
1983.
156.
Ivell, R. 1986.
Biosynthesis of oxytocin in the brain and peripheral
organs.
In: D.
Ganten and D. Pfaff (eds.). Current topics in
Berlin Heidelberg: SpringerVerlag,
neuroendocrinology, Vol. 6.
pp 1-18.
R.,
K.H.
D.
Richter.
1985.
Brackett,
M.J.
Fields and
Ovulation triggers oxytocin gene expression in the bovine ovary.
FEBS Lett. 190:263-267.
Ivell,
R.
and D.
Richter.
The gene for the hypothalamic
1984.
peptide hormone oxytocin is highly expressed in the bovine
corpus luteum:
structure and sequence analysis.
biosynthesis,
EMBO J. 3:2351-2354.
Ivell,
1981.
Niall.
John,M.J.,
Walsh and H.D.
B.W.
Rorjesson,
J.R.
Limited sequence homology between porcine and rat relaxins:
Endocrinology 108:726implications for physiological studies.
729.
Jones, P.B.C.
and A.J.W.
gonadotropinreleasing
Hsueh.
Direct inhibitory effect of
upon luteal luteinizing hormone
rats.
in
hypophysectomized
1980.
hormone
receptor and
steroidogenesis
Endocrinology 107:1930-1936.
of
Endocrinology
Kaltenbach,
1980.
Dunn.
C.C.
and
T.G.
Reproduction in Farm
Reproduction.
In:
Hafez (eds.) .
E.S.E.
Philadelphia. pp 85-113.
Animals.
Lee and Febiger.
Nalbandov.
Niswender and A.V.
Kaltenbach, C.C., J.W. Graber,
G.D.
and
formation
the
on
1968a.
Effect
of
hypophysectomy
82:753Endocrinology
maintenance of corpora lutea in the ewe.
759.
85
Nalbandov.
Niswender and A.V.
Graber, G.D.
Luteotrophic properties of some pituitary hormones in
nonpregnant or pregnant hypophysectomized ewes. Endocrinology
82:818-824.
Kaltenbach,
1968b.
C.C., J.W.
Jackson
Karsch,
Ellicott, D.L. Foster, G.L.
Cook, A.R.
F.J.,
B.
1971.
Failure of infused prolactin to
and A.V.
Nalbandov.
prolong the life span of the corpus luteum of the ewe.
Endocrinology 89:272-275.
Interaction of estradiol and
Convey.
J.S.
1982.
and E.M.
luteinizing hormone releasing hormone on follicle stimulating
J. Anim. Sci. 54:817-821.
hormone release in cattle.
Kesner,
Evidence that
1981.
Anderson.
Kesner, J.S.,
Convey and C.R.
E.M.
in
cattle by
LH
surge
induces the preovulatory
estradiol
increasing pituitary sensitivity to LHRH and then increasing
LHRH release. Endocrinology 108:1386-1391.
on LH,
Rosberg. 1979. Acute suppression of PGF
and S.
epinephrine and fluoride stimulation of adenylate cyclase in rat
J. Cyclic Nucl. Res. 5:55-63.
luteal tissue.
Khan, M.I.
Localization of oxytocin and neurophysin in
KhanDawood, F.S. 1986.
baboon (Papio anubis) corpus luteum by immunocytochemistry.
Acta Endocrinol, 113:570-575,
KhanDawood, F.S. and M.Y. Dawood.
oxytocin.
J.
immunoreactive
Human ovaries
1983.
Endocrinol.
Clin.
contain
Metab.
57:1129-1132.
Presence of oxytocin (OT)
1984.
Dawood.
KhanDawood, F.S. and M.Y.
rabbit and rat.
Sci.
steroid producing glands of the cow,
in
Louis:
Abstr. 31st Ann. Mtg. Soc. Gynecol. Invest. p. 149. St.
CV Mosby.
Paracrine regulation of
1986.
and M.Y.
Dawood.
F.S.
J. Clin. Endocrinol. Metab. 15:171-184.
luteal function.
KhanDawood,
Oxytocin in
1984.
Dawood.
Marut and M.Y.
F.S., E.L.
(Macaca
monkey
cynomolgus
luteum
of
the
corpus
the
fascicularis). Endocrinology 115:570-574.
KhanDawood,
Khar,
KunertRadek and M. Jutisz. 1979. Involvement of
A.,
J.
microtubule and microfilament system in the GnRHinduced release
of gonadotropins by rat anterior pituitary cells in culture.
FEBS Lett. 104:410-414.
Prostaglandin Fla specific
1977.
Kimball, F.A. and L.J.
Wyngarden.
Prostaglandins 13:5t3-564.
binding in equine corpora lutea.
86
Endocrine response
Kittok, R.J., J.H.
Britt and E.M. Convey. 1973.
after GnRH in luteal phase cows and cows with ovarian follicular
cysts.
J.
Anim. Sci. 37:985-989.
Kelly and F. Labrie.
Kledzik,
Auclair, P.A.
G.S.,
L.
Cusan,
C.
hormone
(LH)
1978.
Inhibitory effect of a luteinizing
releasing hormone agonist on rat ovarian LH and follicle
Fertil.
stimulating hormone receptor levels during pregnancy.
Steril. 29:560-564.
Gonadotropinreleasing
1981.
Knecht, M., M.S.
Katz and K.J. Catt.
hormone inhibits cyclic nucleotide accumulation in cultured rat
granulosa cells. J. Biol. Chem. 256:34-36.
Koos,
The large and small cells of the
1981.
Hansel.
ultrastructural
and function differences.
bovine corpus luteum:
Dynamics of
HunzickerDunn (eds.).
In:
N.B.
Schwartz and M.
Ovarian Function.
Raven Press, New York. pp. 197-203.
R.D.
and W.
Effect of
1978.
Saito.
Koyama, T., T. Ohkura, T.
Kumasaka and M.
postovulatory treatment with a luteinizing hormonereleasing
hormone analog on the plasma level of progesterone in women.
Fertil.
Steril. 30:549-552.
Effect of colchicine on in
1971.
J.
and J.V.
Milligan.
vitro ACTH release induced by high K and by hypothalamusstalk
median eminence extract.
Endocrinology 89:408-412.
Kraicer,
J.
Jonis and A.
D.
Schams,
Vullings,
Immunocytochemical demonstration of oxytocin
in bovine ovarian tissues. Acta Endocrinol. 109:537-542.
Kruip,
T.A.M.,
Klarenbeek.
H.G.B.
1985.
1980.
Demonstration
Roy.
Kumari, G.L., S.
Vohra, L.
Joshi and S.
of luteinizing hormone/human chorionic gonadotropin receptor
binding inhibitor in aqueous extracts of frozen human corpus
luteum.
Harm.
Res. 13:57-67.
1964.
Casida.
Collins, W.J. Tyler and L.E.
Labhsetwar, A.P., W.E.
Effect of progesterone and oxytocin on the pituitaryovarian
relationship in heifers. J. Reprod. Fertil. 8:77-83.
LaCroix,
M.C.
and
G.
Kann.
prostaglandinsF2aand E2 in
Comparative studies of
1982.
late cyclic and early pregnant
in vitro synthesis by endometrium and conceptus; effects
sheep:
of in vivo indomethacin treatment on establishment of pregnancy.
Prostaglandins 23:507-526.
Lacy, P.E. 1975.
79:170-187.
Endocrine secretory mechanisims.
Am.
J.
Pathol.
87
Lacy,
Fink.
1968.
P.E.,
S.L.
Howell,
D.A.
Young and C.J.
hypothesis of insulin secretion. Nature 219:1177-1179.
New
Abrogation by
in
AMP
accumulation
prostaglandin F2a of LHstimulated cyclic
Biochem.
Biophys.
Res.
isolated rat corpora lutea of pregnancy.
Comm. 68:1294-1300.
Lahav,
M.,
A.
Freud
and
H.R.
Lindner.
1976.
Chang. 1974. Prostaglandins F and
Lau, I.F., S.K.
Saksena and M.C.
ovulation in mice.
Fertil. 40:467-469.
J.
Reprod.
Storage of neurohypophysial
K.
and K.
Jayasena.
1970.
International
hormones and the mechanism for their release.
Vol
Encyclopedia of Pharmacology and Therapeutics. Section 41,
1.
Pergamon, Oxford. pp. 111-143.
Lederis,
LeMaire,
W.J.
and J.M.
Interrelationships between
1975.
Marsh.
J.
prostaglandins,
cyclic AMP and steroids in ovulation.
Reprod.
Fertil. Suppl. 22:53-74.
Lemon, M. and M.
Loir. 1977. Steroid release in vitro by two luteal
J.
cell types in the corpus luteum of the pregnant sow.
Endocrinol. 72:351-59.
HoYen. 1986.
Zhou and B.
Leung, P.C.K., T. Minegishi, F. Ma, F.
Induction of polyphosphoinositide breakdown in rat corpus luteum
by prostaglandin F2a. Endocrinology 119:12-18.
The role of
J.F.
Morris and G.
Fink.
1985.
C.E.,
microfilaments in the priming effect of LHreleasing hormone: an
Endocrinol. 106:
ultrastructural study using cytochalasin B. J.
Lewis,
211-218.
1982.
Curl.
F.W.
Bazer and J.S.
G.S.,
W.W.
Thatcher,
Metabolism of arachidonic acid in vitro by bovine blastocysts
and endometrium.
Biol. Reprod. 27:431-439.
Lewis,
Fogwell,
Pexton, R.L.
Lewis, G.S., L. Wilson Jr., J.W. Wilks, J.E.
Thayane
and
E.K.
Inskeep.
1977.
S.P.
Ford, R.L.
Butcher, W.V.
PGF
and its metabolites in uterine and jugular venous plasma
2a
J.
Anim.
Sci.
and endometrium of ewes during early pregnancy.
45:320-327.
Lin,
3
binding to
( H)prostaglandins
1977.
and C.V.
Rao.
discrete
for
evidence
dispersed
bovine
cells:
luteal
Res. Comm. 78:510Biophys.
prostaglandin receptors.
Biochem.
M.T.
516.
88
Inhibition of steroidogenesis at
and R.O.
Greep.
1971.
various sites in the biosynthetic pathway in relation to induced
Endocrinology 88:602-607.
ovulation.
Lipner, H.
Liu,
T.C.
1986.
Synthesis and release of
and G.L.
Jackson.
luteinizing hormone by rat anterior pituitary cells: effects of
cytochalasins B and D. Endocrinology 119:236-243.
LuborskyMoore, J.L., K. Wright and H.R. Behrman. 1979. Demonstration
by
of luteal cell membrane receptors for prostaglandin Fe
Med.
Biol.
Exp.
Adv.
ultrastructural
and binding analysis.
112:633-638.
Mackenzie,
An experimental investigation of the mechanism
K.
1911.
of milk secretion with special reference to the action of animal
extracts. Q. J. Exp. Physiol. 4:305-330.
Effect of chronic
Magness, R.R.,
1981.
J.M.
Huie and C.W. Weems.
of
infusion
intrauterine
ipsilateral
or
contralateral
prostaglandin E2 (PGE9) on luteal function of unilaterally
ovariectomized ewes. Prostaglandins Med. 6:389-401.
Haginiwa, A. Nakayama and
K.
Ishii, I.
Nakazawa, K.
Iizuka.
1983.
Detection of immunoreactive human placental
oxytocin and its contractile effect on the uterine muscle.
Endocrinol.
Jpn. 30:389-396.
Makino,
T.,
R.
The ovarian
Mapletoft,
1976.
R.J.,
D.R.
Lapin and 0.J. Ginther.
component of the local pathway between a
artery as the final
gravid uterine horn and ovary in ewes. Biol. Reprod. 15:414-421.
Mapletoft,
PGFa
Effects of
1977.
Ginther.
R.J.,
D.R.
Lapin and 0.J.
Anim.
J.
Sci.
and PGE9 on
corpora lutea of ewes.
45(Suppl. 1):18S.
Prostaglandin formation by the sheep embryo and
1981.
G.J.
as
endometrium
an
indication of maternal recognition of
pregnancy.
Biol. Reprod. 25:56-64.
Marcus,
Addition of colchicinetubulin
Margolis, R.L. and L. Wilson.
1977.
the mechanism of substoichiometric
complex to microtubule ends:
74:
U.S.A.
Sci.
Acad.
colchicine poisoning.
Proc.
Natl.
3466-3470.
Marsh, J.M. 1971. The effect of prostaglandins on the adenyl cyclase
of the bovine corpus luteum. Ann. N.Y. Acad. Sci. 180:416-425.
89
The role of cyclic AMP and
and W.J.
1973.
Lemaire.
In:
N.R.
prostaglandins in the actions of luteinizing hormone.
Academic
Function.
Gonadotropins and Gonadal
Moudgal
(ed).
Press, New York. pp. 376-390.
Marsh,
J.M.
Martal,
J.,
M.C.
LaCroix,
C.
Loudes,
M.
WintenbergerTorres.
Trophoblastin,
1979.
protein present in early pregnancy in sheep.
Saunier and S.
antiluteolysin
J. Reprod. Fertil.
an
56:63-73.
1978.
Rawlings.
Hill and N.C.
Martin, T.E., D.M.
Henricks, J.R.
J.
Active immunization of the cow against oestradio1-175.
Reprod.
Fertil.
53:173-178.
An
1962.
Savard.
Marsh and K.
gonadotropin in vitro. J. Biol. Chem. 237:1801-06.
Mason,
N.R.,
J.M.
Lachance and
Massicotte,
J.,
J.B.
Borgus,
R.
AMP
cyclic
of
hCGinduced
Inhibition
steroidogenesis in rat luteal cells by an
Steroid Biochem. 14:239-242.
action
of
1981.
accumulation and
F.
LHRH
Labrie.
agonist.
J.
Effects of
1986.
Hosoya.
Matsui, H., H.
Yazawa, N. Suzuki and T.
glucocorticoid and cycloheximide on the activity and amount of
235:699J.
RNA polymerase I in nuclei of rat liver. Biochem.
705.
Identification of relaxins in
Matsumoto, D. and W.A.
Chamley. 1980.
porcine follicular fluid and in the ovary of the immature sow.
J.
Reprod.
Fertil. 58:369-375.
Effects of estradio1-175 and
Maurer,
R.A.
Gorski.
1977.
and J.
pimozide on prolactin synthesis in male and female rats.
Endocrinology 101:76-84.
McCracken,
1984.
Update on luteolysisreceptor regulation of
J.A.
Res.
pulsatile secretion of prostaglandin F2m from the uterus.
Reprod. 16:1-2.
Factors
1971.
Goding.
McCracken,
D.T.
Baird and J.R.
J.A.,
affecting the secretion of steroids from the transplanted ovary
Prog. Norm. Res. 27:537-582.
in the sheep.
Rec.
Effects of oxytocin on the oestrous cycle of the
J.A.
Milne,
1963.
Austr.
Vet.
J. 39:51-52.
ewe.
Concurrent uterine venous and
1980.
Milvae,
R.A.
and W.
Hansel.
prostaglandin F concentrations in heifers
ovarian arterial
J. Reprod. Fertil. 60:7-15.
treated with oxytocin.
90
Prolongation of the
Milvae, R.A., B.D. Murphy and W. Hansel. 1984.
bovine estrous cycle with a gonadotropinreleasing hormone
Reprod. 31:664-670.
analog.
Biol.
1982.
Brennecke and R. Webb.
Kraemer, S.P.
Mitchell, M.D., D.L.
Pulsatile
of
oxytocin during the estrous cycle,
release
pregnancy and parturition in sheep. Biol. Reprod. 27:1169-1173.
Macroscopic
1978.
Dott and D.G. Cran.
Moor, R.M., M.F.
Hay, H.M.
identification and steroidogenic function of atretic follicles
in sheep.
J. Endocrinol. 77:309-318.
The corpus luteum of the sheep:
Moor, R.M. and L.E.A.
Rowson. 1966.
functional
relationship between the embryo and the corpus
luteum.
J.
Endocrinol. 34:233-239.
Comparative Morphology of the
Mossman,
1973.
and K.L.
H.W.
Duke.
The University of Wisconsin Press, Madison,
Mammalian Ovary.
Wisconsin. pp. 209-220.
Bohnet and F.A.
H.G.
Tams,
R.
Mukhopadhyay, A.K., A.
Kumar,
Oxytocin and vasopressin have no effect on
1984.
Leidenberger.
progesterone production and cyclic AMP accumulation by rat
luteal cells in vitro. J. Reprod. Fertil. 72:137-141.
Effect of
1982.
Connell.
C.D.
B.M.
Evinson and P.J.
embryo on luteolysis and termination of early pregnancy in sheep
with cloprostenol. Biol. Reprod. 26:263-269.
Nancarrow,
The effect of
Stabenfeldt and C.L. Sauter. 1979.
Neely, D.P., G.H.
J.
Reprod.
exogenous oxytocin on luteal function in mares.
Fertil. 55:303-308.
Chacos and
Manna, N.
Falck, S.
Snyder, J.R.
Involvement of eicosanoids in release of
1985.
J.
Capdevila.
lobe of the rat
oxytocin and vasopressin from the neural
Endocrinology 116:2663-2668.
pituitary.
NegroVilar, A., G.D.
Nett,
W.E.
M.A.
Diekman,
A.M.
Akbar,
T.M.,
R.B.
Staigmiller,
Secretion of prostaglandin
1976.
Ellinwood and G.D. Niswender.
F2c in cycling and pregnant ewes. J. Anim. Sci. 41:144-153.
The effect of
1979.
Rowson.
Booth and L.E.A.
R.,
W.D.
oxytocin treatment on the levels of prostaglandin F in the blood
Reprod. Fertil. 49:17-24.
of heifers. J.
Newcomb,
Placental activity in the mouse in
Beck. 1939.
and N.
Newton, W.H.
the absence of the pituitary gland. J. Endocrinol. 1:65-75.
91
Rat preprorelaxin:
1982.
H.,
Hudson and J. Haley.
P.
complete amino acid sequence derived from cDNA analysis. Ann.
N.Y.
Acad.
Sci. 380:13-21.
Niall,
Wathes,
D.G.
D.C.
Nicholson,
R.W.
Swann,
G.D.
Burford,
H.D.,
oxytocin
of
Porter and B.T.
Pickering.
1984.
Identification
Regul.
the testis and in adrenal tissue.
and vasopressin in
Pept. 8:141-146.
Nett.
1976.
Diekman and T.M.
M.A.
Niswender, G.D., T.J.
Reimers,
Blood flow: a mediator of ovarian function. Biol. Reprod. 14:6481.
Farin and H.R.
C.E.
Niswender,
R.H.
Fitz,
G.D.,
Schwall,
T.A.
Sawyer.
1985.
Regulation of luteal function in domestic
Rec. Prog. Horm. Res. 41:101-151.
ruminants: new concepts.
1981.
Sawyer.
Niswender,
G.D.,
D.E.
Suter and H.R.
J.
regulating receptors for LH on ovine luteal cells.
Fertil. Suppl. 30:183.
O'Byrne, E.M., J.
and
B.G.
Flitcraft,
Steinetz.
Factors
Reprod.
G.
Weiss
Hochman,
Sawyer,
J.
W.K.
and
bioactivity
Relaxin
1978.
immunoactivity in human corpora lutea.
Endocrinology
102:1641-
1644.
Olmsted,
J.B.
and G.G.
Biochem. 42:507-540.
Borisy.
1973.
Microtubules.
O'Shea, J.D., D.G.
1979.
Cran and M.F. Hay.
of the sheep. J.
Anat. 128:239-251.
Ann.
Rev.
The small luteal cell
1986.
Cellular
Wright.
Rodgers and P.J.
J.D.,
R.T.
composition of the sheep corpus luteum in the mid and late
luteal phases of the oestrous cycle. J. Reprod. Fertil. 76:685-
O'Shea,
691.
Intraovarian release of eggs in
Osman,
P.
and J.
Dullaart.
1976.
J.
rat after indomethacin treatment at prooestrus.
the
Reprod.
Fertil. 47:101-103.
Ott,
The galactagogue action of the thymus
I, and J.C.
Scott. 1910.
and corpus luteum. Proc. Soc. Exp. Biol. Med. 8:49.
Ovarian steroids
1982.
Convey.
Padmanabhan, V,
K.
Leung and E.M.
selfpriming effect of luteinizing hormone
modulate
the
in
vitro.
cells
releasing hormone on bovine
pituitary
Endocrinology. 110:717-721.
92
Pant,
1977.
Fitzpatrick.
R.J.
Hopkinson and
C.R.N.
H.C.,
luteinizing hormone
progesterone,
Concentration of oestradiol,
and folliclestimulating hormone in the jugular venous plasma of
J. Endocrinol. 73:247-255.
ewes during the oestrous cycle.
Effects of prostaglandin Fla, on
progesterone production in cultured bovine
agonistinduced
Biol. Reprod. 31:427-435.
luteal cells.
Pate, J.L.
and W.A.
Condon.
1984.
Prostaglandins F
1975.
Weems and E.K. Inskeep.
Pexton, J.E., C.W.
uterine and ovarian venous plasma from nonpregnant and
in
pregnant ewes collected by cannulation. Prostaglandins 9:501509.
Priming effect of
1979.
G.
Fink.
and
A.J.M.C.
luteinizing hormone releasing factcl in vitro: role of protein
Endo
and cyclic AMP. J.
synthesis, contractile elements, Ca
crinol. 81:223-234.
Pickering,
Wolfgang and K.
H.
Konrad,
the corpus
in
hormones
Neurohypophyseal
Lett. 6:1-6.
Neuroendocrinol.
Pitzel,
L.,
W.
1984.
Annemarie.
luteum of the pig.
Baird and
D.T.
Shaw,
R.W.
Bramley,
Currie,
Popkin, R., T.A.
A.
of
luteinizing
hormone
1983.
Specific binding
H.M.
Fraiser.
Biophys.
Biochem.
tissue.
luteal
releasing hormone to human
Commun. 114:750-756.
Res.
Prostaglandin
Powell, W.S., S. Hammarstrom and B. Samuelsson. 1974a.
41:103
Biochem.
Eur. J.
F4t receptor in ovine corpora lutea.
.107.
Localization
Samuelsson. 1976.
Hammerstrom and B.
Powell, W.S., S.
luteum plasma
of a prostaglandin F9a receptor in bovine corpus
Eur. J. Biochem. 61:605-611.
membranes.
1974b.
Samuelsson and B. Sjoberg.
Powell, W.S., S. Hammarstrom, B.
The
Lancet
receptor
in
human
corpora
lutea.
F2a
Prostaglandin
1:1120.
Butcher and E.K.
B.R., R.L.
effect of the conceptus and of
46:784-791.
Pratt,
Rahe,
Inskeep.
PGE2 in
1977.
ewes
Antiluteolytic
J.
Anim.
Sci.
Harms.
Newton and P.G.
Fleeger, H.J.
Owens, J.L.
1980.
Pattern of plasma luteinizing hormone in the cyclic cow:
Endocrinology 107:498dependence upon the period of the cycle.
R.E.
503.
93
in
1971.
Changes
Arimura and A.V. Schally.
responsiveness
luteinizing hormonereleasing
to
hormone (LHRH) in anestrous ewes pretreated with estradiol
benzoate.
Biol.
Reprod. 4:88-92.
Reeves,
J.J.,
A.
pituitary
Magness, J.M. Huie,
Stigler, G.L. Hoyer, R.R.
L.P., J.
Weems.
Huecksteadt, G.L. Whysong, H.R. Behrman and C.W.
PGF13induced luteolysis in
1981.
on
Effect of PG E1 or PGE
2
nonbred ewes. Prostaglandins 21:957-972.
Reynolds,
T.P.
Rice,
Subcellular localization of
1985.
Thorburn.
Physiol.
.J.
Can.
the
luteum.
oxytocin
ovine corpus
Pharmacol. 63:309-314.
G.D.
and
G.E.
in
Lack of direct inhibitory
1985.
Richardson, M.G. and G.M.
Masson.
action of oxytocin on progesterone production by dispersed cells
from human corpus luteum. J. Endocrinol. 104:149-151.
Riley, J.C.M. and J.C. Carlson. 1986. Association of phospholipase A
activity with membrane degeneration during luteolysis in the
rat.
Biol. Reprod. 34(Suppl 1):135.
1976.
Inhibition of hCGinduced
R.H.
and E.S. Johnson.
ovarian and uterine weight augmentation in the immature rat by
Exp. Biol. Med. 152:432-436.
Proc. Soc.
analogs of GnRH.
Rippel,
Bahr.
Khan and J.M.
Calvo, I.M.
Ritzhaupt, L.K., R.A. Nowak, F.O.
during
luteum
corpus
1986.
Adenylate cyclase activity of the
J. Reprod. Fertil. 78:361-366.
the oestrous cycle of the pig.
prostaglandin F2a
1976.
Does
McCracken.
released from the uterus by oxytocin mediate the oxytocic action
of oxytocin? Biol. Reprod. 15:457-463.
Roberts,
J.S.
and J.A.
Roberts, J.S., J.A.
McCracken, J.E.
Gavagan and M.S.
Soloff. 1976.
Oxytocinstimulated release of prostaglandin F9a from ovine
in
vivo:
correlation with estrous cycle and
endometrium
Endocrinology 99:1107-1114.
oxytocinreceptor binding.
H.H.
In:
Reproduction in cattle.
1977.
Cupps (eds.). Reproduction in Domestic Animals.
Press, New York. pp. 433-454.
Robinson,
T.J.
P.T.
Rodger,
L.D.
and
F.
Stormshak.
hormoneinduced alteration of
Biol.
Reprod. 35:149-156.
1986.
bovine
Cole and
Academic
Gonadotropinreleasing
corpus
luteum
function.
94
Purification, morphology, and
Rodgers, R.J.
O'Shea. 1982.
and J.D.
progesterone production and content of three cell types isolated
J.
Biol.
Sci.
Austr.
from the corpus luteum of the sheep.
35:441-455.
Flint and E.L.
Findlay, A.P.F.
Rodgers,
R.J.,
J.D.
O'Shea,
J.K.
Sheldrick.
1983.
Large luteal cells the source of luteal
Endocrinology 113:2302-2304.
oxytocin in the sheep.
Rondell,
P.
1970.
29:1875-1879.
Rothchild,
Rec.
Follicular
processes in ovulation.
Fed.
Proc.
The regulation of the mammalian corpus luteum.
Prog. Horm. Res. 37:183-298.
I.
1981.
The influence of embryonic
on the lifespan of
tissue homogenates infused into the uterus,
the corpus luteum in the sheep. J. Reprod. Fertil. 13:511-516.
Rowson,
L.E.A.
and
R.M.
Moor.
1967.
Thorneycroft and B.V.
Scaramuzzi,
Tillson,
I.H.
S.A.
R.J.,
estrogen
Caldwell.
1971.
Action of exogenous progesterone and
on behavioral
estrus and luteinizing hormone levels in the
Endocrinology 88:1184-1189.
ovariectomized ewe.
Presence
Schaeffer, J.M., J. Liu, A.J.W. Hsueh and S.C.C. Yen. 1984.
of oxytocin and arginine vasopressin in human ovary, oviduct and
follicular fluid.
J. Clin. Endocrinol. Metab. 59:970-973.
The action of animal extracts
Schafer, E.A. and K. Mackenzie.
1911.
on milk secretion. Proc. R. Soc. Lond. B. 84:16-22.
Schams, B. Bullermann and D.L. Walters. 1984.
Schallenberger, E., D.
ovarian steroids and
Pulsatile secretion of gonadotrophins,
ovarian oxytocin during prostaglandininduced regression of the
J. Reprod. Fertil. 71:493-501.
corpus luteum in the cow.
III.
Oxytocin determination by radioimmunoassay.
D.
1983.
Improvement to subpicogram sensitivity and application to blood
levels in cyclic cattle. Acta Endocrinol. 103:180-183
Schams,
Schams, D., T.A.M.
Kruip and R. Koll. 1985a. Oxytocin determination
steroid producing tissues and in vitro production in ovarian
in
follicles.
Acta Endocrinol. 109:530-536.
Oxytocin
1982.
Glatzel.
D.,
LahlouKassi and P.
A.
concentrations in peripheral blood during the estrous cycle and
after ovariectomy in two breeds of sheep with low and high
fecundity.
J. Endocrinol. 92:9-13.
Schams,
95
Schams, D., S. Prokopp and D. Barth. 1983. The effect of active and
passive immunization against oxytocin on ovarian cyclicity in
Acta Endocrinol. 103:337-344.
ewes.
Evidence for
1985b.
Schams, D., E. Schallenberger and J.J. Legros.
the secretion of immunoreactive neurophysin I in addition to
73:165Fertil.
Reprod.
oxytocin from the ovary in cattle. J.
171.
Oxytocin
1979.
Kruse.
and V.
and
I.
Method
in
cattle.
radioimmunoassay
determination by
Acta
Endocrinol.
92:258-270.
preliminary physiological data.
Schams,
D.,
B.
SchmidtPolex
Evolution, relaxin
1982.
Schwabe, C., L.K.
Gowan and J.W.
Reinig.
Sci. 380:6-12.
Acad.
and insulin: a new perspective. Ann. N.Y.
Segaloff, J.K. McDonald,
Steinetz, G. Weiss, A.
1978.
Carierp and L. Goldsmith.
Hochman,
O'Byrne, J.
E.
B.
Relaxin.
Horm. Res. 34:123-199.
Rec. Prog.
Schwabe,
C.,
B.
Flint. 1981. Circulating concentrations
Sheldrick, E.L.
and A.P.F.
of oxytocin during the estrous cycle and early pregnancy in
Prostaglandins 22:631-636
sheep.
Ovarian oxytocin and luteal
Sheldrick, E.L. and A.P.F. Flint. 1983a.
function in the early pregnant sheep. Anim. Reprod. Sci. 10:101113.
Luteal concentrations of
1983b.
Flint.
Sheldrick, E.L.
and A.P.F.
Reprod.
J.
oxytocin decline during early pregnancy in the ewe.
Fertil. 68:477-480.
Flint.
Mitchell and A.P.F.
Sheldrick,
E.L.,
M.D.
luteal
regression in ewes immunized against
Fertil. 59:37-42.
Reprod.
Delayed
J.
oxytocin.
1980.
1979.
Hansel.
Ayalon and W.
N.
M.,
F.
Milaguir,
Shemesh,
and prostaglandin synthesis by cultured bovine
Steroidogenesis
Reprod. Fertil. 56:181-185.
blastocysts.
J.
1975.
Dzuik.
Bevier and P.J.
G.W.
Chang,
0.D.,
C.C.
Radioimmunoassay of plasma relaxin levels throughout pregnancy
and at parturition in the pig. Endocrinology 97:834-838.
Sherwood,
1974.
O'Byrne.
E.M.
and
Sherwood,
0.D.
characterization of porcine relaxin. Arch.
160:185-196.
and
Purification
Biophys.
Biochem.
96
Christensen and R.A.
D.S.
Howland, R.D. Randel,
Anim.
J.
Bellows.
1973.
Induced LH release in spayed cows.
Sci. 37:551-557.
Short, R.E., B.E.
1981.
Concentrations
Inskeep.
Silvia, W.J., J.S.
Ottobre and E.K.
F2a and 6ketoprostaglandin Fla in the
E2,
of prostaglandins
uteroovarian venous plasma of early pregnant and cycling ewes.
Biol.
Reprod. 24(Suppl. 1):99A.
Concentrations
1984.
Inskeep.
Silvia, W.J., J.S. Ottobre and E.K.
F9a
and
6ketoprostaglandin
Fla in the
of prostaglandins E2,
of
nonpregnant
and
early
pregnant
uteroovarian venous plasma
Biol.
Reprod. 30:936-944.
ewes.
Studies on the growth and
1971.
Robertson.
and H.A.
Smeaton, T.C.
J.
atresia of Graafian follicles in the ovary of the sheep.
Reprod.
Fertil. 25:243-252.
The role of hormones in the
1958.
Nalbandov.
and A.V.
Smith, J.S.
Am.
relaxation of the uterine portion of the cervix in swine.
J.
Res. 19:15-18.
Vet.
Statistical Methods, 7th ed.
Snedecor, G.W. and W.G. Cochran. 1980.
Iowa State University Press, Ames, Iowa.
The
1969.
Snook, R.B., M.A.
Brunner, R.R. Saatman and W. Hansel.
effect of antisera to bovine LH in hysterectomized and intact
Reprod. 1:49-58.
heifers.
Biol.
O'Byrne and R.L. Kroc. 1980.
Dilatation
Stubblefield (eds.).
and P.G.
Raven Press, New York. pp. 157-177.
Cervix.
Steinetz, B.G.
E.M.
Naftolin
In: F.
of the Uterine
ZelinskiWooten and S.E. Abdelgadir. 1987.
F.,
M.B.
Stormshak,
Comparative aspects of the regulation of corpus luteum function
Dhindsa (eds.).
Mahesh and D.S.
In:
V.B.
in various species.
Co.
Regulation of Ovarian and Testicular Function. Plenum Publ.
New York. (In Press).
Disparate effects
1979.
Stouffer, R.L., W.E. Nixon and G.D. Hodgen.
gonadotropinstimulated
and
basal
on
of
prostaglandins
progesterone production by luteal cells isolated from rhesus
Biol. Reprod.
monkeys during the menstrual cycle and pregnancy.
20:897-903.
Studies on the role of
1976.
Beers.
S.
and W.H.
Strickland,
vitro response of
In
plasminogen activator in ovulation.
and
nucleotides,
cyclic
granulosa cells to gonadotropins,
prostaglandins. J. Biol. Chem. 251:5694-5702.
97
Wathes,
D.G.
D.C.
Birkett,
Swann, R.W., P.L. O'Shaughnessy, S.D.
Biosynthesis of oxytocin in
Pickering.
1984.
Porter, and P.T.
the corpus luteum. FEBS Lett. 174:262-266.
Adenylate cyclase activity of
avian granulosa: effect of gonadotropinreleasing hormone. Gen.
Endocrinol. 48:515-524.
Comp.
Takats, A.
Tan,
Tan,
and F.
Hertelendy.
1982.
Effect
1982a.
Biggs.
Tweedale and J.S.G.
oxytocin on the bovine corpus luteum of early pregnancy.
Fertil. 66:75-78.
Reprod.
G.J.S.,
R.
of
J.
Oxytocin may
1982b.
Biggs.
G.J.S.,
R.
Tweedale and J.S.G.
J.
of the human corpus luteum.
play a role in the control
Endocrinol. 95:65-70.
Bazer, R.M.
Knickerbocker, F.F. Bartol, F.W.
Thatcher,
W.W., J.J.
Maternal recognition of pregnancy
Drost.
1985.
Roberts and M.
relation to the survival of transferred embryos: endocrine
in
aspects. Theriogenology 23:129-144.
Are follicular maturation and oocyte maturation
1977.
Thibault,
C.
independent processes? J. Reprod. Fertil. 51:1-15.
Preovulatory and
1975.
Gerard and Y. Menezo.
Thibault,
M.
C.,
Fertil.
Reprod.
J.
ovulatory mechanisms in oocyte maturation.
45:605-610.
Restall and W.
B.J.
Currie,
W.B.
Prostaglandin F concentration in the utero
J.
ovarian venous plasma of the ewe during the oestrous cycle.
Endocrinol. 53:325-326.
Thorburn,
G.D.,
R.I.
1972.
Schneider.
Cox,
Thorburn,
R.I.
G.D.,
1973.
Schneider.
Cox,
Currie,
W.B.
B.J.
Restall
and
W.
and progesterone concentra
Prostaglandin
tions in the uteroovarian venous plasma of the ewe during the
oestrous cycle
18:151-158.
F
and
pregnancy.
J.
Reprod.
Fertil.
Suppl.
An inhibitory influence of
granulosa cells and follicular fluid upon porcine oocyte meiosis
Endocrinolgy 96:922-927.
in vitro.
Tsafriri,
A.
and C.
Charming.
1975.
Varying response to luteinizing
1979.
Leymarie.
Ursely, J.
and P.
hormone of two cell types isolated from bovine corpus luteum.
Endocrinol. 83:303-310.
J.
Concentration of relaxin in the blood
Wada, H. and M.
Yuhara. 1961.
Silver
Proc.
serum of pregnant cow and cow with ovarian cyst.
Jubilee, Kioto University, Kioto, Japan. p. 61.
98
Pulsatile secretion of
1984.
Schallenberger.
Walters, D.L. and E.
gonadotrophins, ovarian steroids and ovarian oxytocin during the
periovulatory phase of the oestrous cycle in the cow. J.
Fertil. 71:503-512.
Reprod.
1983.
Walters, D.L., D. Schams, B. Bullermann and E. Schallenberger.
and
steroids
ovarian
Pulsatile secretion of gonadotropins,
Reprod.
Biol.
the cow.
ovarian oxytocin during luteolysis in
28(Suppl 1):142.
Pulsatile
1984.
Walters,
D.
Schams and E. Schallenberger.
D.L.,
ovarian steroids and ovarian
secretion of gonadotrophins,
oxytocin during the luteal phase of the oestrous cycle in the
J. Reprod. Fertil. 71:479-491.
cow.
Wang,
S.C. and G.S. Greenwald. 1985. Effect of cycloheximide
peptide and
injected at proestrus on ovarian protein synthesis,
Biol.
and ovulation in the hamster.
steroid hormone levels,
Reprod. 33:201-211.
Physiological inhibitor of and from the ovary.
Ward, D.N. 1981.
pursuit
In
(eds.).
and
H.J.
Vogel
G.
Jagiello
New York Academic Press,
Bioregulators of Reproduction.
York. pp. 371-387.
Wathes, D.C.
Swann. 1982.
and R.W.
Nature 297:225-227.
In:
of
New
Is oxytocin an ovarian hormone?
Porter and B.T.
D.G.
Birkett,
Wathes,
D.C.,
R.W.
Swann,
S.D.
Characterization of oxytocin, vasopressin and
Pickering. 1983a.
Endocrinology
from the bovine corpus luteum.
neurophysin
113:693-698.
Porter and
Drife, D.G.
Hull, J.0.
Wathes, D.C., R.W. Swann, M.G.R.
sources of the posterior
Gonadal
B.T.
Pickering.
1983b.
Prog. Brain Res. 60:513-520.
pituitary hormones.
Variations in
1984.
Swann and B.T. Pickering.
Wathes,
D.C., R.W.
vasopressin and neurophysin concentrations in the
oxytocin,
J.
bovine ovary during the oestrous cycle and pregnancy.
Fertil. 71:551-557.
Reprod.
Hull
Porter, M.G.R.
Pickering, D.G.
human
Neurohypophysial hormones in the
Drife.
1982.
and J.0.
Lancet II: 410-412.
ovary.
Wathes, D.C., R.W. Swann, B.T.
1986.
Pickering.
Porter and B.T.
Swann, D.G.
D.C.,
R.W.
Pfaff
D.
In:
Ganten and D.
Oxytocin as an ovarian hormone.
Berlin
6.
Current Topics in Neuroendocrinology, Vol.
(eds.).
Heidelberg: SpringerVerlag, pp 129-152.
Wathes,
99
Webb,
1981.
Falconer and J.S. Robinson.
J.
Mitchell,
relationships between peripheral plasma concentrations
Temporal
progesterone and 13,14dihydroketo prostaglandin
of oxytocin,
F
during the oestrous cycle and early pregnancy in the ewe.
21
Prostaglandins 22:443-454
R.,
Weiss,
M.D.
G.,
E.M.
product of
194:948-949.
Steinetz.
O'Byrne and B.G.
the human corpus luteum of
a
Relaxin:
Science
pregnancy.
1976.
Dierschke and
D.J.
Steinetz,
G.,
B.G.
Relaxin secretion in the Rhesus monkey. Biol.
Weiss,
G.Fritz.
Reprod.
1981.
24:565
567.
Prostaglandin F,a
1986.
Davis.
Weakland and J.S.
West, L.A., L.L.
1,4,t
inositol
and
stimulates phosphoinositide hydrolysis
in
isolated
bovine
luteal
cells.
triphosphate (IP) synthesis
Biol. Reprod. 34(Suppl 1):138.
The effect of oxytocin on the corpus luteum of the
Wilks, J.W. 1983.
Contraception 28:267-272.
monkey.
Paracrine regulation of the
Behrman. 1983.
Williams, A.T. and H.R.
ovary by gonadotropin releasing hormone and other peptides.
Endocrinol. 1:269-277.
Reprod.
Sem.
Wolff. 1970. Possible role of microtubules in
Williams, J.A. and J.
67: 1901U.S.A.
Sci.
Acad.
Natl.
thyroid secretion. Proc.
1908.
Grisham and K.M.
L.M.
Mizel,
S.B.
Bamburg,
Wilson,
L.,
J.R.
Interaction
of
drugs
with
microtubule
proteins.
Creswell. 1974.
Fed. Proced. 33:158-166.
Prostaglandin
Butcher and E.K. Inskeep. 1972.
Wilson, L., Jr., R.L.
F a in the uterus of ewes during early pregnancy. Prostaglandins
2
1:479-482.
Yang,
Prostaglandin
1973.
LeMaire.
Marsh and W.J.
J.M.
N.S.T.,
in
rabbit Graafian
by ovulatory stimuli
induced
changes
Prostaglandins 4:395The effect of indomethacin.
follicles.
404.
Yang,
Corpus luteum LH
1981.
Yen.
Neira and N.H.
P. Franchimont and
In:
(LHRBI).
inhibitor
binding
receptor
of Reproduction.
Regulation
Intragonadal
C.P. Channing (eds.).
Academic Press, New York. pp. 133-166.
K.P.,
E.S.
100
Zarrow,
M.X.,
G.M.
1956.
Bullard.
following
72:260-264.
Neher,
Dilataion
treatment with
D.
Breenen and J.F.
D.M.
Sikes,
of the uterine cervix of the sow
Obstet.
Gynecol.
J.
relaxin. Am.