BIOLOGY
OF
46, 1001-1006
REPRODUCTION
Evidence K1YOSHI
OKUDA,3
for Oxytocin
A1UO
Department
(1992)
MIYAMOTO,
of Physiology,
Receptors HELGA
Technical
in Cultured
SAUERWEIN,
University
Bovine J.
FLORIAN
of Munich,
8050
Luteal
Cells1
SCHWEIGERT,
and
DIETER
Freising-WeihenNtephan,
SCHAMS2
Germany
ABSTRACT Specific
receptors
at different using the placement
(Days
of
different
binding
K
2.6
=
OT
We luteal
site
8-12,
on intact luteal cells 15-18 of the estrous
and
OT
was
that
plays
paracrine
M
an
and
The
an apparent
K, of 4.9
at the
maximal
action
important
5.9
K, of the X
are
stage
on
the
role
physiological
(Days
in
the
well
role
during feedback
established
of the
peptide
that
oxytocin
(UT)
is mediated
in the
control
of luteal
to its involvement release of luteal
in luteolysis, progesterone
investigated with dispensed cells in there are still no clear indications affect progesterone release from 20]. Recently, it has been clearly stimulates acutely the progesterone tissue in a micnodialysis system in
after
mid-,
and
MATERIALS
lected
from
Accepted
CL from
a local
January
the
many species. However, whether or not UT does luteal cells in vitro [15demonstrated that UT release of bovine luteal vitro [21]. This action of
Forschungsgemein.schaft. of Science
AM.
3Permanent
of one
two
DNA.
versus
two
cell culture and therefore was observed at all luteal
supporting
the
hypothesis
other
luteal
functions
AND
luteal
that in
a
buffer. were
into
blood
were
col-
within
10-20
mm
after
in part
was supported
by the
Moninaga
by the Japan
Ilohshikai Society
and for
of Animal
Science,
Faculty
8-12,
and
Cell Preparation
mucus). of the
and
cells
The luteal
Days
of three
15-18 CL were
procedure [22]
was
as described used
for
the
for
the
ovary
arteries
for
and
to loosen
the
cells. The laboratory
perfusion
for
pen ovary). 15 mm,
of
mm)
were
as described
between
the
vas-
were subsequently in ice-cold EGTA
then placed on rate of 5 mI/mm
two capillaries EGTA buffer
iso-
ovaries were permM EGTA, 10 mM 7.4) to remove vas-
connection ovaries submerged
the
dissociation
a filter unit and for each outlet
Ovaries followed
were perby a wash-
buffer with calcium (10 mM HEPES, 140 mM NaCI, 7.1 mM KC1, 5.0 mM CaCl2; pH 7.4) for 15 mm. Final dissociation was obtained by perfusion with the wash-buffer (300 ml)
when
Deutsche
observed
the Promotion
of Agriculture,
Days
experiment,
The ovaries were perfused at a flow
(generally fused with cows
3-5,
each
perfusion
cular endothelial transported to the
stages.
Fleckvieh
connective tissue, and assigned to three stages Days
For
defined by macroscopic and CL) and the uterus
CL with several adaptations and modifications. Teflon micnocapillaries (internal diameter, 0.3
cular
METHODS
German
Luteal stages were of the ovaries (follicles
of hepatocytes
containing with the
Division
Japan
Observation
6)
=
fmol/.tg
below. Immediately after slaughter the fused with an ice-cold EGTA buffer (0.1 HEPES, 140 mM NaCl, 7.1 mM KCI; p1-I
gassed during large
FAX: 08161-714404.
address:
Okavama,
(n
0.73
was
and/or
Dissociation
inserted
USFS).
2Correspondence. University,
late
slaughterhouse
21. 1991. supported
demonstrated in diswith an association
experiments
for the of OT
receptor,
representing
lation
22, 1992.
Received November ‘This research was
OT
release
A two-step
a direct has been
of CL with
the
ovulation.
Tissue
would be mediated via specific UT receptors. The present study was thus conducted to investigate the presence of specific binding sites for UT in cultured bovine
Ovaries
DNA.
(CI.) assay
pooled.
OT
(‘ollection
by
progesterone
phase,
function
evidence for the systemic comes from reports that luteal regression in sheep
early,
site
capacity
CL used binding
(size, colon, consistency, CL were accordingly
corpus luteum (CL) in many spedata for ruminants support a sys-
taglandin F2a [11, 12]. The best role of UT in luteal regression immunization against UT delays
from
of
exsanguination. observation
is synthe-
establishment of a positive UT and endometnial pros-
CL cells
was
of the
its
lutca
corpora a radioreceptor
8-12).
regulation
regression through the loop between luteal
[13, 14]. In addition effect of UT on the
by
50%
iO’#{176} M’;
individual Significant
bovine
specificity
in
X
from
receptors
of a binding
of 8.8 finol/Lg
state of the population.
CL
bovine
OT
Binding presence
the 2.6
cells
for
Additionally, was
a capacity
and
nor to the OT receptor
mid-luteal of OT
W’
site
Cultured
examined
fashion.
sized and secreted by the cies [1-10]. The available temic
were
DNA.
fmol/ag
INTRODUCTION It is now
demonstrated.
cycle)
high-affinity
10
to the assay conditions variation within the
a direct
or autocrine
of
a capacity
observed.
neither individual
binding
conclude
iO
were had
sites related to reflect
stages.
X
Sites
low-affinity
binding appeared
3-5,
1251-labeled OT antagonist ld(CH2)5,Tyr(Me)2, Thr4,Tyr.NH291-vasotocin. studies with various related peptides. Scatchard analysis revealed
constant The
(OT)
oxytocm
for
stages
Okavama
700.
1001
the
0.05% collagenase
collagenase solution
tissue
showed
usually
after
with 5% perfusion. petri dish
and was
a mushy 30
mm.
0.1% BSA. The perfusion recycled and terminated appearance,
All perfusion
which buffers
CO2 in 95% 02 before pH adjustment The treated tissue was transferred containing Medium 199 (with Earle’s
was were and to a salts
1002
OKUDA
and 25 mM 1-IEPES). Luteal CL matrix with steel combs.
cells were Dissociated
CL were
for 10 mm
pooled
taining (DNase
and
0.05% I), and
stirred
collagenase, 0.1% BSA,
ester meshes (200 undissociated tissue times by centnifugation
0.005% and then
dispersed CL cells
from the from three
in Medium
Reagents
199 con-
Deoxyribonuclease filtered through
I polv-
m, 150 rim, and 80 p.m) to remove fragments. The filtrate was washed three for 5 mm at 50 X g with the culture
tamed cell or fibrocytes inary
suspension (about
contained 20%), and
experiments
cells
was
Cell blue
(n
estimated
6) the
=
[23]
no
at the
viability exclusion.
higher The oh-
very few endothelial cells envthrocvtes. In prelim-
DNA
content
beginning
culture. There was no significant per well, indicating a constant DNA content in the preparation means of cell number, assuming
was
of the
and
after
luteal 48
and
in
ii
difference in DNA content number of cells. Thus the was routinely estimated by 1.5 X 10 cells per 1 ig
of I)NA.
3-lipotropin, penicillin, were purchased from
Laboratories, Inc. Hoechst (Frankfurt,
Statistical
viable maintained
cells/well/mI, in 24-well
Data on were
ligand analyzed
[26] The
using initial
nonlinear parameters
[28] and were sum of squares selected model
binding with
tocin method
was [24]. The
The
then iteratively was minimized. was assayed
criteria
were
based
for
the cells Mg2-free The
three times with Hanks’ Balanced
cells
were
then
incubated
with
30000
the presence of 0-100 nM unlabeled UT. were performed in 0.5 ml mHBSS containing and 0.1% BSA for 3 h at room temperature.
cpm
and 7.5). in
I2s10f
All incuhations 5 mM MgCl2 Equilibrium
centrauon binding
of the
was
neuropeptide ment 1-10)
Binding
The
incubation
ml
I M NaOII,
was
terminated
by rapid
(fragwas
washing
of
ml’IBSS containing 0.1% BSA. After three were removed from the plates with 0.5 and
the
hound
radioactivity
was
counted.
for the
radioneceptor
assay
on bovine
lu-
affinity MgCI7
used
Mg2’
on Mn2 curves
of unlabeled UT ligand was similar ions
for
(Fig.
AVP
2, A and
were
B). In contrast,
was considerably lower in the in the presence of MnCI,. Therefore,
in the
following
and OVT in in the pres-
different:
AVP
the
relative
presence of only MgC12
experiments.
Qaracteristics
Figure 3 shows the various related peptides tagonist
cells
The potency ‘25I-labeled
the
of than
The
reason,
of 5 mM appeared to be superior with regard to specificity for UT compared to MnCl2 at the same
displacement
cells with ice-cold washes, the cells
t-
further assays were perper well. MgCl2 at a con-
the
tested.
[26].
from early, by Student’s
ml (Fig. IB). For this formed with 400000-500000
ence
nM)
values
to cell preparations were compared
was established in the to 500000 cells per well/
reactivity of [Arg8]-vasopressmn (AVP), [d(CHTyr(UMe)2, Unn8]-vasotocin (UT-antagonist), I d( CH,)5,D-Tyr( UEt )Z,Vall, C1t81-vasopressmn (OT/AVP-antagonist), desgh’cinamide (Mg8)vasopnessin (Des-glv-AVP), LHRH, morphine-modulating and of porcmne--lipotropin concentrations (0-640
a particular
probability
cell numbers. A linear relationship amount of binding from 100000
concentration. displacing
(MMNP), different
on accepting
calculated
Conditions
conditions
was reached under these conditions. No significant tracer degradation was observed during the incubation as determined by tnichloracetic acid precipitation [25]. The cross-
at
[27]. analysis
teal cells as described above were initially validated. It was confirmed that maximal binding was reached after 3 h at 20#{176}C (Fig. IA). Specific binding increased with increasing
lactoperoxidase 48 h by washing
0.5 ml of modified Ca2-free Salts Solution (mHBSS, pH
to luteal program
refined until the weighted The goodness of fit for the by the “nun’s test”: different
rejecting
on the
of t21..O\rF luteal stages
Assay
The
iodinated by a modified culture was stopped after
of UT, OVT, and AVP the LIGAND computer
RESULTS
[d( CH, ),[Tyr(Me)]2,Thr’,Tyn-NI-L9]-vaso-
(OVT)
by
test.
Assays
antagonist
donated
models, i.e. one- or two-site models, were compared using F-test statistics to determine whether a change in the model resulted in a significant reduction of the weighted sum of
Receptor
The
kindly
iterative curve-fitting procedures were calculated by Scatchard
(Coster, Cambridge, MA) for up to 48 h in a humidified atmosphere of 5% CO2 in air at 37.5#{176}C. The culture medium was changed 24 h after the start of the culture.
Radioreceptor
OT/AVP-antagonist, Despurchased from Peninsula
Analysis
Specific binding mid-, and late
prepared culture plates
and amphotericin-B Co. (Deisenhofen,
(Belmont, GA). OT was Germany).
cells
model About 400000-500000 as described above, were
streptomycin, Sigma Chemical
OVF, AVP, UT-antagonist, LHRH, and MMNP were
squares.
C’ell C’ulture
Hormones
Medium 199 with Earle’s salts and 25 mM HEPES, Dulhecco’s modified Eagle’s/Nutrient mixture Ham’s F-12 Medium, mHBSS, HEPES, EGTA, collagenase, DNase, porcine-
Germany). glv-AVP,
medium (Dulbecco’s Modified Eagle’s Medium and Ham’s F-12 medium, 1:1 (vol:vol) supplemented with 10% calf serum, 60 ig/ml penicillin, 100 g/ml streptomvcin, 2 g/ ml amphotericin-B, and 2 mM L-glutamine). The cells were counted with a hemoctometen. than 85% as assessed by trvpan
ET AL.
binding showed
was
displacement on CL cells
highly competition
specific
curves of I2sIU from the mid-luteal for
comparable
UT.
Only to that
the
with stage. UT-an-
of UT.
In
OXYTOCIN
IN BOVINE
RECEPTORS
Ct
Ct
8
.
8
.
.0
1003
B
12
12
CELLS
LUTEAL
.0
(no 0.51
2
4
20
lime
FIG.
1.
Relationship
(h)
between
the
on bovine CL cells from the mid-luteal the absence of 100 nM 01 was used added.
comparison,
an approximately
of the UT/VP displacement
that of UT displaced the other peptides Scatchard linear (Fig. a mean 3). The pM.
Related
higher
plots of the binding 4A). Analysis with the
data (t2tIUVT/OT) LIGAND program
were showed
of 2.6 X of this binding
i09 site
(Ka)
to the
DNA
content
was
5.89
fmol/g
±
0.24 (B)
of the
i09 W’ was 38.89 X
CL cells,
the
±
(n = 8.86
receptor
Scatchand
plots
were
(Fig. 4B). A two-site-model was superior in dethese binding data as analyzed with the LIGAND (p s 0.001). The apparent affinity of the highbinding site (Kat) was 2.62 X #{231}JtO ± 2.4 X 10t0 M_t;
the capacity low-affinity was and
(B,,,1) site, Ka2 8.78 ± curvilinear
was was 2.22
0.73 4.94
0.05 10
± X
fmol/g plots was
not
features
of the
X
DNA. The dependent
ence of either Mg2 on Mn2 during any other assay condition and could morphological
fmol/g 2.30
±
the not
individual
DNA. For the 10 M’ and
appearance on the
incubation be related CL used
of presor on to any
for
cell
were
used.
Table
1 presents on cultured
different
luteal
cells of binding
the
a comparison luteal cells
stages.
The
mid-luteal detectable
was
Nonspecific
binding
Stages
to validate prepared from
A
the assay mid-luteal
Mn
and CL
preparations
only
binding
in the
of
cow
ical
cell
culture
activity
low specific stage cells.
early
and
assays
with
60 40
20
20 0.039
study
[31].
and
been
sheep
found
we
With
used
late
late
luteal
[29, 30].
CL
stages
0.156
OT,
OVT
and
AVP
in the
While
to be dependent
an iodinated
UT on the
receptor activity
this
UT
0.625
ligand
we
found
#{149}OT
2.5
presence
antagonist,
as ligand. This UVI’ ligand that can he t261 without losing its biolog-
10
Peptide concentration unlabeled
the
evidence for the existence of luteal cells. Thus far the UT in ruminant CL membrane
Mg
(nM)
of ‘25l-OVT and mid-luteal stage.
on
of the CL [29], UT binding in the sheep only in CL of pregnant animals [30]. In
B
40
binding from the
seen
and 52%, respectively) than mid-luteal stage (17-27%). the binding sites by Scat-
8O
FIG. 2. Competitive (B) on bovine CL cells
binding of CL of three
was
100
60
concentration
3-
only luteal
cows
has
developmental stage has been observed
#{149} AVP
Peptide
ex-
binding
Our study presents strong UT binding sites on bovine receptor has been demonstrated
80 0
was (Days
DISCUSSION
#{149}OT
100
specific from
chard analysis in the cells of the early and due to the low amount of specific binding..
Speqfic
Luteal
highest
early
stage, whereas in early on late in the
t2Id(CH,)s[Tw(Me)2ThrTyrNH9]O\rf is a highly selective UT labeled to a high specific
at D[ferent
investigation
of the prepared
cells was markedly higher (48% in assays with CL cells from the It was not possible to characterize
our Binding
the
from CL of the luteal stages.
isolation.
In all experiments performed characterize the binding, cells
Later
to cell preparations late (Days 15-18)
of
DNA.
curvilinear
8-12)
tended 5) and
than
None
In 3 of 6 experiments,
B,2 linear
100-fold ‘251-UVT binding. any displacement.
concentration observed scribing program affinity
(Days
concentration
of the showed
affinity capacity
ml
of ‘25l-0’ir and (A) incubation time at 20CC or 4CC, or (B) cell numbers stage. The difference in the binding of 125IOVT bound in the presence and in to calculate the specific binding, expressed as a percentage of total radioactivity
higher
approximately 50% tested
well/
binding
antagonist was necessary to achieve a 50% of the labeled ligand. As shown in Figure 2,
in a concentration
AVP
20-fold
no. xl O/
Cell
of Mg2
40
100
(nM) (A) or Mn2
binding
char-
1004
OKUDA
ET
AL.
0
100
*
-
0
*
0
*
80
o
0
*
*
-
A
OVT
#{149} OT #{163} OT ANT
60-
-
OTNP-ANT #{149} AVP LH RH #{176} Des-gly-AVP o
4020
-
-
I
I
I
#{149}t
I
I
I
I
0 0.039
0.156
0.625
2.5 Peptide
FIG. 3.
binding of ‘25JOjand unlabeled on bovine CL cells from the mid-luteal other abbreviations are given in the text.
Competitive
thetic analogues antagonists. All
concentration
receptor
from
the
bovine
uterus
[32],
the affinity described by Meyer and vine endometrium [33]. In addition to the single binding
but
higher
coworkers site,
for other
two
than the
bo-
classes
of binding states were observed as documented by curvilinear Scatchard plots as well as by the superior fit of a twosite model (LIGAND program). These additional binding states might he attributed to differentially activated forms of the
receptor.
affinity
states
system
of metal
It has been postulated that the different of the UT receptor are related to a complex ion
binding
to different
ceptor molecule [34]. Furthermore, populations have been reported have flO conclusive different binding
regions
re-
several UT binding for uterine cells [35].
site We
explanation for the occurrence furms that were randomly observed
0.089
of the in CL
1000
=
hormones as well as some OT-antagonist; OT/VP-ANT
cells from the mid-luteal phase. served predominance of specific
of their syn= OT/AVP
We assume that the obaffinity states for UT in
specific CL cell preparations might be due to an individual shift within the receptor population. There has been only one investigation of the UT receptor in the bovine CL in which 3H-OT-binding to membrane preparations was found predominantly at the late luteal stage [29]. The Ka of 3.9 x 10 reported was similar to our finding for the we observed
single was
the mid-luteal rations at the is not possible used.
of the
160
(nM)
neurohypophysial stage. 01-ANT
acteristics on cultured bovine luteal cells comparable to those described for classical UT target tissue such as the uterus. The affinity of the single binding site (linear Scatchard plots, Fig. 4A) was in the same range as that demonstrated for the UT
40
10
The
reactivity UVT
the
site. However, about 10-fold
activity AVP
A
0.057
of the
differed.
showed a higher luteal cell culture
atively pure preparations
capacity of the binding on cultured cells from
stage than on cells from membrane late luteal stage. A direct comparison because of the differences in the
specific with
the higher
ligands
Compared
as well to the
with regard to cell type, whereas of luteal tissue contain numerous
B
0.060
0.048 #{149} #{149} 0 0 LL
S
#{149}
0.045
S
V C
C
0.030
0.019
0.015
0.010
0.000
8.28
16.6 Bound
cells
0.038 0.029
V
FIG. 4. Representative from the mid-luteal
24.9
0.000
33.1
16.5
49.6
66.1
Bound (pM)
(pM)
Scatchard stage.
plots
for
the
competitive
binding
as the H-OT,
cross125j
selectivity for UT binding. Moreover, system in the present study was rel-
S
0.074
prepaof data methods
of #{176}51-OVT and
unlabeled
OT on bovine
CL
membrane cell types.
OXYTOCIN TABLE and
1. late
Specific
luteal
binding
stages
of 01
(mean
on bovine
CL cells
RECEPTORS
from
early,
mid-,
IN
BOVINE
2. Flint
SD).
±
LUTEAL
APF,
glandin.
Days 8-12 (n=11)
(n=5) 1.8 The
10.8
1.71
±
Days 15-18
difference
of 100 nM 01 was used to calculate percentage
of total
*significantly
radioactivity
different
the specific
added,
from
1.2
all other
per
and
in the
binding,
5 x
stages
corpora
4. Wathes
DC,
oxvtocin,
0.73
±
iO
absence
expressed
5, Ivell
as a
cells.
(p -z 0.001)
1983;
highly
expressed
quence
analysis.
6. Walters
DL,
no
information
UT
at all.
represent in luteal available
If this
approximately tissue [36].
50 to 60% of the There is, however,
as to whether
were
the
case,
UT receptor concentrations brane preparations.
endothelial
a different
would
be
cells pattern
expected
clearly
demonstrated release of bovine
that UT stimCL in a mi-
crodialysis system [21]. As in the case of other peptide hormones, the effect of UT requires the presence of specific receptors. Our results reveal the existence of specific bindsites
for UT
in specific
UT
parallels the sue. During
on cultured binding
bovine
to luteal
changes luteal
luteal
cells
cells.
The
at different
in the concentration development UT
its own receptor UT concentrations
of UT in CL tisconcentrations in-
cause a down-regulation of the lier study in the microdialysis
receptor. system
on stimulation stage [211. This
continuously vation appears
late the
hormonal
responses
concentrations.
are
explanations
dent changes in the characteristics of cell activity (e.g. intracellular use of different systems (luteal alysis thesize
of
CL tissue the receptor
speculative. In conclusion, ceptors studies
role
pieces), under
such
with
evidence
with the suggested in the
in a paracrine
to
for
existence
effects of UT observed that luteal UT plays
regulation
and/or
the
of the
autocrine
bovine
We
Frankfurt. Germany.
for the supply
18. Pitsel on
227.
Swann
RW. Is oxvtocin
22.
Kardalinou prolactin
II gene
Brattlehoro F., Scheit in porcine
its
Biomedical;
Fertil
1980:
59:37-42.
effect
in luteal
and
play
II-!.
Enthe
Clinical
and
317-334. in ewes
passive
Endocrinol
At-ta
rela-
during
Oxvtocin.
regres.sion
of active ma\’
oxvtocin
oxvtocin
1985:
luteal
and
B, Breitinger
H. Ovarian
AG (ed.s.),
in ewes.
source
154:756-763.
for ovarian
Delayed
Oxvtocin
immu-
immunization
1982;
a role
102:337-344.
in the control
of the
1982; 95:65-70.
JSG. Effects of oxvtocin
on the bovine
Fenil 1982; 66:75-78. in the cow and sheep.
H. Wuttke
J Reprod
corpus
Fertil
luteum
Suppl
1989;
and vasopressin
W. Inhibitory effect of oxvtocin
porcine
luteal
MC.
cells
Masson
GM. by
celLs. Endocrinology
1988;
122;1’80-
11G. Leidenberger
progesterone
Lack
production
J Reprod
in vitro.
of direct
dispersed
Fenil
from
cyclic
AMP
ac-
72:137-141.
action
human
FA. Oxvtocin
and
1984;
inhibitory
cells
D. Oxvtocin
corpus
LR, Kirsch
DG,
luteum. Lokse
metabolism
in suspensions
pared
the
from
R, Bohnet
on
of oxvtocin
on pro-
luteum.
J Endo-
corpus
104:149-151. A. Schams
Laharca
stimulates Biol
CL, Carlson and
caudate
progesterone
Reprod
GP, Doody
primary
process
1991;
from
l.A. Wisniewski
cultures
of bovine
release
JA. Xenobiotic
of isolated
liver.
micro-
44:1163-1170.
Am J Vet
hepatucytes Res
pre.
1986;
47:2043-
C. Paigen
and
sensitive
DNA
Anal
Biochem
procedure.
and
V. Oxvtocin
preliminary
determination
physiological
data,
h’
radioimmu-
Acta
F.ndocrinol
92:258-270.
Ban.skota
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