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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