Release of immunoreactive oxytocin and neurophysin I by cultured luteinizing bovine granulosa cells

Release of immunoreactive

_oxytocin

and

_neurophysin

I

by

cultured

_luteinizing

bovine

_granulosa

cells

V.Geenen,

_{J.J. Legros,}

M.T.

_{Haz\l=e'\e-Hagelstein,}

F. Louis-Kohn,

_M.J.

Lecomte-Yerna,

A.Demoulin and P. Franchimont

Laboratoire de

_{Radioimmunologie,}

Unité de

Neuroendocrinologie1,

Centre

_Hospitalier

Universitaire_B23,Universitéde

_Liège

-Sart

Tilman,

B 4000

_Liège

and

Département

de

_Gynécologie

et

_{d'Obstétrique2,}

Maternitéde

_l'Hôpital

de

Bavière,

Boulevard de la_{Constitution,}B 4020

_Liège,

_Belgique

Abstract.

We

_investigated

the

_production

of

_oxytocin

(OT)

and

_{oxytocin-neurophysin}

(bNpI) by

bovine granu-losa cells cultured in presence of10%foetal calfserum,a

condition knowntoinduce

spontaneous

luteinizationof these cells. The

_production

of immunoreactive OT was

significantly higher

in the cultures of

granulosa

cells harvested from

_large

follicles thaninthose derived from small follicles.

Chromatography

on

Sephadex

_G-25

showed similar elution sites of ovarian and

synthetic

OT, while

_{high performance}

liquid chromatography

revealed

two

_peaks

of

_{OT-immunoreactivity,}

oneof which

(\m=+-\

_65%

of the total

immunoreactivity)

coincided with

_synthetic

OT. In another

_experiment,

we_{could observe}a

gradual

increase of

OT,

bNp

Iand _progesterone

_{production by}

granulosa

cells derived from

_large

follicles,

in relation with the incubation time. The mean _{molar ratio OT:}

bNp

I was _2.2\m=+-\0.5

(sem),

and we found a

significant

positive

correlation between the

_production

ofOT and

bNp

I

(r

=

0.77;

P_<

0.01)

and between the

_production

of OT and

progesterone (r

=

0.80;

P<

0.01).

Further-more, the cellular OT and

bNp

I content of

_large

follicles-derived

_granulosa

cells before culturewas 4\p=n-\5

times lower than the total amount of OT and

bNp

I

produced during

a72-h

incubation,

suggesting

anactive

synthesis

of these

_peptides.

These data show that bovine

granulosa

cells areableto

produce

OT and

_bNp

I,

probably by

an active

biosyn-thesisas_{observed in the}

hypothalamo-neurohypophysial

systemand that the

granulosa productions

ofOT,

bNp

I

and_progesteroneare

closely

_related.

Oxytocin

(OT)

has been shown

to

be

_present

inthe

corpus luteum of the

_sheep

(Wathes

& Swann

1982),

cow

(Fields

et al.

1983;

Wathes et al.

1983)

and human

_(Wathes

et al.

1982;

Khan-Dawood &

Dawood

1983).

The

identification

of the

_specific

OT carrier

_protein,

bovine

_neurophysin

I

_(bNp

_I),

has also been

_reported

inextracts

of bovine

_corpus

luteum

_(Wathes

etal.

_1983).

The OT

_gene

is

highly

expressed

in the bovine corpus luteum

during

the

mid-luteal

_phase

of

the oestrous

cycle

(Ivell

&

Richter

₁₉₈₄₎

and studies

_using

_{incorporation}

of

[35S]cysteine

show that

isolated

ovine and bovine

luteal cells

can

synthétise

OT

by

_{way of}

_precursor

protein

similarto that found in the

hypothalamus

(Swann

et

al.

_1984).

The function of

ovarian OT is still rather un¬

clear;

some

observations

_suggest

that OT could

intervene

in

the

_luteolytic

process

either

_by

exert¬

ing

an

inhibitory

effect

on

the

_progesterone

_syn¬

thesis

_(Tan

et

al.

_1982a,b)

or

by stimulating

the

release of the

_luteolytic

_{prostaglandin}

F2a

(PGF2a)

from

the

uterus

(Roberts

et al.

1976);

PGF2a,

in turn,

induces the

secretion of ovarian OT

(Flint

&

Sheldrick

1982),

so that OT and

PGF2a

would

engage

in a

positive

feedback

loop leading

to

luteolysis.

However,

OT could

_play

another role

in

Requests

for

_reprints:

Vincent

Geenen,

Unité de

Neuroendocrinologie,

Labo¬ ratoire de

_{Radioimmunologie,}

Université de

_Liège

-Sart

Tilman,

CHU-B23,

B4000

_Liège,

_Belgique.

the control of the

oestrous

_cycle

_{as, in} cows and

sheep,

the

_highest

values of

_circulating

OT levels

and

amounts

of

ovarian extracts are found in the

early

and

mid-luteal

_phases

_(Schams

et al.

1982,

1983;

Wathes

et

al.

1984),

and as the

peripheral

OT levels

increase

in

_synchrony

with

_changes

in the

progesterone

production

butstart to decline

_again

shortly

before

_progesterone

levels decrease at

luteolysis

(Schams

etal.

1983).

We have

_judged

of interestto

_study

the secretion

of the OT-related

_{neurohypophysial peptides by}

cultured bovine

_granulosa

cells

_undergoing

luteini-zation

and

to

investigate

the

relationship

with the

increase of the

_progesterone

_production

observed in

such

conditions.

Material and Methods

Cultures

_of

bovine

_{granulosa-cells}

The

_technique

has been described in detail before

(Hen¬

derson 8c Moon

1979;

Henderson &Franchimont

1981).

Bovine ovaries were obtained from a local

slaughter¬

house and carriedtothe

laboratory

inchilledminimum

essential medium

(modified)

with Earle's salts

(EMEM),

supplemented

with HEPES buffer

₍₂₀

mM),

glutamine

(2 mM),

penicillin

(50

IU/ml),

streptomycin

(50

_pg/ml),

amphotericin

(0.625

pg/ml)

and non-essential amino

acids. Ovarian folliclesweredissected and the

granulosa

cells

_scraped

with a

platinum loop

into chilled _EMEM,

supplemented

as _{above. The}

_pooled

cellswere washed three times with the medium and cell

_clumps

were

dissociated with

gentle

mechanical

_agitation

and re¬

peated

aspiration

with a

glass pipette;

an

aliquot

was

taken to determine total cell number

using

a

haemocy-tometer and the mean

_percentage

of viable cells deter¬

mined with

_nigrosin

dye

approached

to _45%. 10s 'live cells' were_{cultured in multi-welled tissue culture dishes}

at37°Cin 1.0 mlculturemedium

_consisting

of10%

(v/v)

foetal calf serum and 90% EMEM

supplemented

as

above,

but without HEPES buffer. The

gas-phase

was5%

C02:95%

air. After a_24-h

period

_{necessary for adher¬} ence _{of viable}

cells,

_{the medium} was _{removed and}

replaced by

freshone.

Ina _first

experiment, granulosa

cells were harvested from

_large

(>

10mmin

diameter)

and small

(<

10mmin

diameter)

follicles,

and cultured for 48 h after the medium

replacement.

Afterincubation,themediumwas

collected and stored at

—

20°C before further

analyses

(hormone

assays,

chromatography

on

Sephadex

_G-25

and

_{high performance liquid chromatography).}

In a second

_experiment,

we

performed

cultures of

granulosa

cells collected from

large

follicles

_only;

after the medium

replacement,

the cultures

(total

number=

12)

were divided in three groupsof four

replicate

cul¬

turesandwere_{incubated for24,}48or72h.Themedia

werethen collected and storeduntil_{assays ofOT,}

bNp

I

and _{progesterone.} At the end of

incubation,

the cells

were washed and stained with

haematoxylin

and

eosin;

the

granulosa

cells formed a

monolayer

and

_appeared

healthy;

morphological

aspect of luteinizationwas indi¬

cated

by

cellular

hyperplasia

and

hypertrophy

with an

increase in the

cytoplasm:nucleus

ratio.Five

_samples

of 10s viable non-incubated

granulosa

cells collected from

large

follicleswere submitted to

lysis

in 1.0ml distilled

water in order to determine the initial OT and

_bNp

I

cellularcontent.

Radioimmunoassay

ofOT

The antiserum was _raised

against

OT

coupled

to

thyro¬

globulin

asdescribed

by

_Robinson

(1980):

_{2 mgsynthetic}

OT

(Sandoz,

Basel,

Switzerland

—

462

IU/mg)

were

conjugated

to5_{mg bovine}

_{thyroglobulin (Sigma,}

_{type 1)}

in 3.0 mlwater,

using

7 mg

l-(3-dimethylaminopropyl)-3-ethylcarbodiimide

(Aldrich,

_Europe).

After 18 h at

room

_temperature,

_{7 mg}

_{hydroxylamine-HCl}

wasadded

and the mixturewas_{incubated for5}hatroom_tempera¬

ture. The

antibody

was

produced

in female rabbits

by

injection

of 0.5 mg

OT-thyroglobulin

mixture in 1.0ml water, emulsified in an

equal

volume of

complete

Freund's

adjuvant.

Rabbits were

injected eight

times

monthly

into

_{multiple paravertebral}

intradermal sites and

_bleeding

occurred

_monthly

after the fifth immuniza¬ tion.The antiseraweretitrated in serial dilutions

against

radioiodinatedOTand the dilution

_binding

30—40%of

thetracerwasused in

preparing

the assay.

OTwas labelled with 12SI

according

tothe chloramine

method

(Greenwood

et al.

₁₉₆₃₎

and

purified

on a

DEAE-Sephadex

A-25 column

(0.8

x7

cm)

as

_previously

described

_(Legros

Sc Franchimont

_1971).

Mean

_specific

activity

of radioiodinated OT

ranged

from 200 to550

mCi/mg.

The omission of the reduction_step

_by

metabi-sulfite did not

improve

_the

immunoreactivity

nor the

specific

activity

of the tracer. The

radioimmunoassay

procedure

was carried out in0.05M ammonium bicar¬

bonate

buffer,

pH

7.5,

supplemented

with ovalbumin

(4

_g/1),

Na2-EDTA

(7

g/1),

tween20

(5

_ml/1)

and

trasylol

(50000

IU/1).

The best antiserum

(As02,

finaldilution 1:

9000)

was _{incubated for} 4

_days

at 4°C with 0.1 ml

[125I]OT

(±600()CPM)

and 0.1 ml various amounts of

synthetic

OT or

samples

to be

_{assayed. Separation}

of

bound and free

antigen

was

performed by

double anti¬

body

precipitation

of boundOT.The standardcurvefor

radioimmunoassay

of OT is shownin

_Fig.

1. The lowest

detectable value of OTwas5

_pg/tube

and the

_intercept

of

50%

relative

binding

on _the curve

corresponded

to

140 pg

synthetic

OT. There was an

incomplete

cross-reaction with

_{arginine-vasotocin}

(AVT),

whilecross-reac¬

tions for other

peptides

when

_compared

toOTata_B/B„

displacement

of_50%

_(on

a

weight

basis)

were:

30 10 10 20 50 100 200 pg /tube 5 10 20 ng/tube bNpI Pro-Leu-Gly-NH2

Fig.

1.

Standardcurvefor the

radioimmunoassay

of

oxytocin

_(OT)

and cross-reactioncurveswith

arginine-vasotocin

(AVT),

arginine-vasopressin

(AVP),

bovine

oxytocin-neurophysin

I

_(bNp

I),

terminal

_tripeptide

of OT

_{(Pro-Leu-Gly-NH2),}

bovine follicle

stimulating

hormone

(FSH)

and

_luteinizing

hormone

(LH).

0.41%;

OT terminal

_{tripeptide (Pro-Leu-Gly-NH2),}

1.25%;

follicle

_stimulating

hormone

(FSH)

and luteiniz¬

ing

hormone

(LH),

< _{0.003%. Validation of the radio¬}

immunoassay

was _assessed

by

themeasurement ofOT

concentrations after addition of knownamounts_{of syn¬}

thetic OT into the culture medium

(EMEM)

containing

10%

foetal calfserum or_not;no

significant

interference

could be observed and theOTcontentof non-incubated culture medium

supplemented

with 10% foetal calf

serum _{was below the limit} of detection. Therefore,

a _{direct assay of OT in unextracted culture medium} was allowed with a

sensitivity

of5

_pg/tube

(50

pg/ml

medium).

The

_intra-assay

coefficient of variation

_ranged

from 4.5 to8.5%

_along

thecurve, and the

inter-assay

coeffi¬

cient,

_{assayed by}

replicate

estimate of thesame5medium

samples

within different

radioimmunoassays,

was 13.5%.

Radioimmunoassay

of bNp

I

The antiserum

against

_bNp

I wasraisedin arabbit and

the cross-reaction with bovine

_{vasopressin-neurophysin}

II

_(bNp

II)

was < _{0.5% and other bovine anterior}

pituitary

hormones<0.1%.The

_{radioimmunoassay}

_pro¬ cedure has been described in detail elsewhere

by

Legros

(1976).

Radioimmunoassay

of

progesterone

Antiserum

P04/11

_Tg

was

kindly supplied by

Prof.

Umberto Rosa

(Consiglio

delle

Riserche, Pisa,

Italy)

and

usedata_{final dilution of 1:5000;}tracerwas

[125I]proge-sterone

(IRE, Fleurus,

Belgium), conjugated

as

11-succi-nyl-tyrosine.

The _percentagecross-reaction of the

anti-serum _in

comparison

of_progesterone

(100%)

was 0.3%

for

_{17a-hydroxyprogesterone,}

0.05% for

_pregnenolone

and <_0.001% for

20a-hydroxy-4-pregnen-3-one,

corti¬

costerone,

cortisol,

5a-dihydrotestosterone,

4-andro-stene-3,17-dione,

testosterone and

_{oestradiol-17ß.}

The limit of the detection was 0.05

ng/ml. Separation

of

bound and free hormoneswas

performed

with

dextran-coated charcoal

(0.1%

dextran

T-70,

1%neutral charcoal in albumin

_phosphate

buffer,

_pH

7.4);

centrifugation

was

made after 15 min and

_{radioactivity}

of the

_pellet

_(free

hormone)

wascounted.

Chromatography

Five

_samples

(0.4

ml

_each)

of incubated media were

pooled

from cultures of

large

follicles-derived

_granulosa

cells and were

applied

to the _top ofa column

(0.9

X

27

cm)

of

Sephadex

G-25,

equilibrated

with0.1 formic acid,

pH

2.3, and eluted witha flow-rate of 1 ml/min. Fractions

_{(1.0 ml)}

were_{collected and}

lyophilized;

_later,

they

were_{redissolved in 0.2 ml}

radioimmunoassay

buffer

and

assayed

for OT in

_duplicate.

A similar chromato¬

graphy

was_carriedouttwice in thesameconditions and

the material from the main

peak

of

OT-immunoreacti-vity

(fractions

13

—

18)

was

pooled

and

lyophilized

_sepa¬

rately

before

high

performance

liquid chromatography

(HPLC)

analysis.

The HPLC _system consisted of a Waters Associates Model 6000 A solvent

delivery

system,

equipped

witha

model 480 LC

_{Spectrophotometer,}

a Model_{720 system}

controller anda _{Model 330}UV/V¡Sabsorbance detector.

The column was a _{Waters Associates 10 µ}

Bondapak

C-18

(0.4

x30

cm).

The eluent consisted of1% trifluoro-aceticacid anda_linear

gradient

_{of 0}to60%acetonitrile

was used with a _duration of 90 min at a flow-rate of

1

ml/min.

Fractions of 1.0mlwere

collected,

lyophilized

and

_assayed

for OT in

duplicate.

Statistical

_analysL·

All values are

given

as mean±SEM. When OT values

were below the limit of

detection,

they

were nottaken

intoaccount,and their numberwasmentioned.Thedata

from the first

_experiment

were

_subjected

to

_{analysis by}

unpaired

Student'sr-test.

Thecoefficent of linear correlation wascalculated

by

themethodof the least_squares.

Results

As indicated in Table

1,

immunoreactive OT could

be detected in the medium

aftera

48-h incubation

of bovine

_granulosa

cells

harvested

from

_large

follicles,

and

was

undetectable

only

in

2out of 20

cultures.

The OT

and

_progesterone

_productions

were

significantly

lower

in the

cultures

of granu¬

losa cells from small

follicles,

and

OT-immunore-activity

wasnot

detected

in 12outof

20

cultures.

Table1.

Oxytocin

(OT)

and

progesterone

production by

cultured bovine

_granulosa

cells,

obtained from

large

or small

follicles.

Origin

of

_granulosa

cells

Large

follicles

(>

10mm

_diam.)

Small follicles

(<

10mm

diam.)

Oxytocin

(OT)

(pg/105

cells/48

h)

Undetectable

Progesterone

(ng/10s

cells/48

h)

Undetectable 83±4.5*

2/20

133 ± 6**

0/20

66.2±3.2 12/20 72±4

0/20

Valuesare

_given

asmean±SEMofdetectable values.

*f<0.05

(t

=

1.85;

df

=24),

**P_<1 10"9

(t

=

8.26;

df=

38)

compared

to the

corresponding

values in the

smallfollicles-derivedcultures

_(unpaired

Student's

i-test).

Fig.

2 shows the

_Sephadex

G-25 elution

_profile

of

_{OT-immunoreactivity}

contained

in 2.0 ml of

pooled

media collected from

5 cultures of

_large

follicles-derived

_granulosa

cells.

The main

_peak

appeared

in the

same

position

as

standard

OT;

it was

preceded by

a

smaller

peak

in

fraction

12,

and some

OT-immunoreactivity

was

detected

_up to

fraction

18. The amount of OT eluted corre¬

sponded

to

±95%

of the

total

OTcontent meas¬

ured

in

the

_pooled

media.

HPLC

_analyses

of the material contained

in

frac¬

tions 13—18

eluted from two

_separate

Sephadex

G-25

_{chromatographies}

are

shown

in

Fig.

3. Two

peaks

of

_{OT-immunoreactivity}

wereeluted in each

run;

the second

peak

was

co-eluted

with standard

OT and

_corresponded

to ± 65

%

of the total

OT-immunoreactivity

eluted.

An

OT-like

immunore¬

activity

(±

35%)

was

detected

in eluates

collected

at

19—20 min of the

_system,

but

itwas not

co-eluted

with

standard

_preparations

of

AVTorAVP.

As

indicated

in

_Fig.

4,

we

could

observea

gradual

increase of

the

_granulosa

cell

_production

of

OT,

bNp

I

and

_progesterone

in

relation

with the dura¬

tion of

the cultures.

The mean molar ratio OT:

bNp

Iwas

2.2

±

0.5;

therewas a

significant positive

relationship

between the

_production

of

OT and

bNp

I

(r

=

0.77;

<

0.01)

and

between the

production

of OT

and

_progesterone

_(r

=

0.80;

<

_0.01).

The

initial cellular

OT and

_bNp

I con¬

tent

of

large

follicles-derived

granulosa

cells was

59

±

5.4

_pg/105

cells and 0.49

± 0.05

_ng/105

cells,

respectively.

This was 4—5

times

lower

than the

total

amount of OT

and

bNp

I

_{produced during}

the

72-h

incubation

_{(respectively}

295 ±

_pg/105

cells

and

2.22 ±0.11

_ng/105

_cells).

Discussion

When

bovine

_granulosa

cells

are cultured in

medium

_containing

10%

foetal calf

serum,

they

undergo morphological

and functional modifica¬

tions indicative of

a

_spontaneous

luteinization

(Henderson

& Moon

_1979).

Our data demonstrate

that,

in

the

same

conditions,

luteinizing

bovine

granulosa

cells release immunoreactive

OT

and

bNp

I

in the culture medium.

A

_significant

differ¬

ence

_{appears between}

the

productions

of

OT

and

progesterone

by granulosa

cells from

_large

or

small

follicles,

_suggesting

an

influence of

the

_stage

of

OT OH1' 70 60 50 >- ¿0 o 30 · 20 Vo

I

15 20 ELUATE(mi) 25

Flg.

2.

Sephadex

G-25 column

(0.9

x ₂₇

_cm)

_{chromatography}

of incubated media

_pooled

from cultures of

large

follicles-derived

granulosa

cells. Maximal elution sites of

_synthetic

and iodinated OTare

represented.

Each eluted

fraction

(1.0 ml)

was

lyophilized,

redissolved in 0.2 ml buffer and

assayed

forOT in

duplicate,

sothelimitofdetection

was

brought

to10

pg/ml.

.-, 60 ¿0 -o ₃₀ 20 o oc 10 AVP OT - - -r AVT 40 C£ LU O 20 < 10 20 30 Í0 50 60 70 80 90

ELUTION TIME (min.)

Fig.

3.

HPLC

profiles

ofthe

OT-immunoreactivity

contained in the main

_peak

_{(fractions 13-18)}

eluted fromtwo _separate

Sephadex

G-25

_{chromatographies.}

Each eluted

_{(1.0 ml)}

fraction was

lyophilized,

_redissolved in0.2 ml buffer and

assayed

for OT in

_duplicate,

sothe limitof_detectionwas

brought

to10

_pg/ml.

The

_position

of standard

_peptides

OT

(Sandoz, Switzerland),

AVTandAVP

_(UCB

i

i

m

Is

i

I

\

\

'D

Fig·.

4.

Production of

OT,

bNp

Iandprogesterone

(expressed

in

_ng/105

cells,mean±semfor4

_replicate

cultures)

by

bovine

_granulosa

cellsin function of theindicatedincubation time.

nature of

OT

produced

by

cultured

_granulosa

cells,

_gel

filtration

_{chromatography}

reveals similar

elution

_profiles

of ovarian

OT and

_synthetic

OT,

while

HPLC

_analyses

evidence

some

heterogeneity

of the

_granulosa

cell

_product,

65%

of the

OT-immunoreactivity

reflecting

the authentic hor¬ mone. As

the other

peak

of

OT-immunoreactivity

derives from the

material

contained

in

fractions

13—18 from G-25

_{chromatography,}

the molecular

weight

must

be close

tothe OTone

and, thus,

this

peak

does

not

_represent

some

larger

_precursor.

This unknown

_peptide

is not one

of

the other

neurohypophysial peptides,

AVPor

AVT,

asindi¬

cated

_by

HPLC

studies,

and

we

hypothesize

that it

may

represent

eithera

degradation product

of OT or a

small

intermediary

form

of

the OT

synthetic

pathway.

In

each

case,the

presence

of this OT-like

immunoreactive material

can

partially explain

a

molar ration

_OT:bNp

I

_slightly

_higher

than the

theoretical

one

(1:1).

We

think that

the

observed

_findings

reflect an

active

_biosynthesis

of the OT-related

_{peptides by}

ovarian cells

rather thana

passive

release

of these

hormones,

and different

_experimental

_{facts argue}

for

an

active

synthetic

_process

analogous

to the

hypothalamo-neurohypophysial

pathway by

cleav¬

age from

a common

_precursor

(Land

et al.

1983):

first,

the

_parallel

and

correlated

_productions

of OT

and

_bNp

1 with a

molar

ratio rather

close

to

the

theoretical

one

and,

mainly,

the

_gradual

culture

medium enrichment

in OT and

_bNp

I

_along

the

incubation

time,

with

a total amount of

OT

and

bNp

I

_produced

4

—

5 times

_greater

than

the initial

cellular

content

of these

peptides.

Otherwise,

direct

evidence for

OT

_{synthesis by}

_{corpus luteum has}

been

_{provided by}

two

other groups

(Ivell

&

Richter

1984;

Swann et

al.

1984)

but there

have been

no

previous

reports

referring

to

granulosa

cells.

Another

_aspect

of

our

findings

deserves

some

discussion. In

a

preliminary

_report

(Geenen

et al.

1984),

we

showed

a

significant

negative

correlation

between the

_productions

of

_neurophysins

and

_pro¬

gesterone

by

cultured

_granulosa

cells

from

_large

follicles,

and

a

negative

but

not

significant

correla¬

tion

between the

_productions

of OT

and

_progeste¬

rone.

This

discrepancy

with the

_present

results

could be

due

to

the

fact that we in

the

previous

experiment

have used

a

radioimmunoassay

detect¬

ing

all

the immunoreactive

forms of

_{neurophysins,}

including

vasopressin-neurophysin,

and that we

have considered

_only

one

period

of incubation

and

not successive

incubation

timesas in this work. In

the

_light

of the

_present

_data,

it

_{appears that}

the

release

_and,

_probably,

the

_biosynthesis

of OT and

bNp

I

_by

cultured bovine

_granulosa

cellsare

closely

there-fore,

to

the luteinization

of

these

cells.

Other

authors have

_reported

that

OT

could

influence

the

steroidogenesis

of bovine corpus

luteum;

small

doses

of OT

(4—40 mIU/ml,

corresponding

to

8-80

_ng/ml

as 1

pIU

=±

2 pg

OT),

stimulated the

production

of

_progesterone

while

_higher

OTcon¬

centrations

_{(800 mIU/ml)}

_{inhibited the basal pro¬}

gesterone

production

and

the

response

of

luteal

cells

to

hCG

(Tan

et al.

1982a).

In ewesand cows,

circulating

OT levels

increase

in

_parallel

with

the

progesterone

concentrations,

and the

_highest

val¬ ues are

found in

the

early

and

midluteal

phases

(Schams

et

al.

1982;

Schams

1983).

The

_present

study

is

in

accordance with

these

_reports

and we

think that the cultures of

_granulosa

cells

constitute

a

suitable

approach

to

_investigate

more

profoundly

the ovarian function

of OT

and

the

_paracrine

rela¬

tionship

between

OT and the

_gonadal

steroido¬

genesis,

especially during

the

_{early phases}

of lutein¬

ization.

_Recently,

itwas

reported

that the neuro¬

hypophysial

peptides

AVT,

AVP and OT

exerted

a

direct

_stimulatory

effect on the

biosynthesis

of

pregnenolone

and

_progesterone

_by

cultured rat

testicular

cells

_(Adashi

et

al.

1984).

A

similar

me¬

chanism could

occur in

the

_{ovary and could be}

postulated

on

the basis

of

the

positive

correlation

observed

in

this

_study

between

the

_production

of OT and

_progesterone

_by

cultured

_luteinizing

granulosa

cells.

Acknowledgments

We wishtothankMrs. Y.Heinemann andV.Lechanteur who

_typed

the

manuscript

and Mr.

_J.

Collette who drew all

_graphs.

Dr. V. Geenen is

Aspirant

du Fonds National de la Recherche

Scientifique

de

_Belgique.

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