The dynamics of the testosterone response of perifused mouse Leydig cells to hCG and Arginine Vasopressin

Vol. 146, No. 1, 1987 July 15, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 216-223

THE DYNAMICS OF THE TESTOSTERONE RESPONSE OF PERIFUSED MOUSE LEYDIG CELLS TO hCG AND ARGININE

A. Tahri-Joutei,

M.T.

INSERM U. 123 Bld Received

March

23,

166,

Latreille,

and G. Pointis

Maternite

de Port-Royal,

VASOPRESSIN

Baudelocque,

75014

Paris,

France

1987

The effect of hCG and Arginine-Vasopressin (AVP) on testosterone production by purified mouse Leydig cells was examined under dynamic conditions in a perifusion sytem. A rapid and dose-dependent increase in testosterone release was induced by a 5 min exposure of the cells to increasing concentrations of hCG (0.01 to 1 rig/ml). The testosterone response to hCG was Gaussian in distribution with -9 peak value by 100 min. A 12 h pretreatment of Leydig cells with 10 M AVP enhanced testosterone accumulation in the perifusate under basal conditions, but markedly reduced the hCG-stimulated testosterone production. The basal and hCG-stimulated testosterone secretion profiles by freshly isolated Leydig cells were, however, unaffected by the continuous presence of the same dose of AVP. These results support the finding that AVP acts directly on Leydig cells. They support the hypothesis of a possible role of neurohypophysial peptides on reproductive functions in the mouse by modulating steroidogenesis at the testicular level. 17 1987 Academic Press, Inc.

It

has

peptide the

reproductive

physial

of pituitary

in

adrenal

species

circulating

testis

zation

of

specific

0006-291X/87

the

These

the

human.

itself.

that These

AVP receptor

(5)

large

this

peptide

observations,

216

Recently,

of are

effects

on

from

the

result

the neurohypo-

(4,6,7)

deficient

could

(8)

pineal

identi-

from

several

vasopressin-like

together cells

the

have been

testes

amount which

in Leydig

to

and oxytocin and

of

inhibitory

appeared

rats

$1.50

0 I987 by Academic Press, Inc. of reproduction in any form reserved.

exerts

by AVT (2).

ovaries

of Brattleboro indicated

male,

(AVP)

The

administration

effects

gonadotropins

(3,4),

AVP (8) the

(1).

glands

testes

within

to

Arginine-Vasopressin

including in the

that

reported (AVT)

function

hormones

fied

Copyright AN rights

previously

Arginine-Vasotocin

suppression

found

been

peptide

in pituitary

be synthesized with suggested

the

and locally

characteri-

that

these

Vol. 146, No. 1, 1987

BIOCHEMICAL

neurohypophysial normal pure

animal. mouse

effect fied

peptides Recently Leydig

we have

cells

of AVP on basal mouse Leydig

might

(10).

AND BIOPHYSICAL

exert

some

established In

the

and hCG-stimulated

cells

was examined

physiologic

an in vitro present

dynamic

function perifusion

investigation,

testosterone under

RESEARCH COMMUNICATIONS

production conditions

in

the

system the

of

direct

by purias a function

of time. MATERIALS

AND METHODS

MATERIALS. Human chorionic gonadotropin (hCG, 9000 IU/mg) and Arginine' vasopressin (AVP) were provided respectively by Boehringer, Mannheim France and Calbiochem (San Diego, CA, U.S.A.). Bovine serum albumin (BSA) and Collagenase type I (144 u/mg) were from ICN Pharmaceuticals Inc. (Cleveland, OH, U.S.A.), Glucose, Deoxyribonuclease I (DNA-ase I) from Sigma Chemical Co (St Louis, MO, U.S.A.). L-Glutamine, Sodium bicarbonate, N- hydroxyethylpiperazine-N'ethanesulfonic acid (Hepes) and medium 169 were obtained France). from Eurobio Bio-Gel P2 (200-400 mesh) and Percoll were ?Paris, purchased respectively from Bio-Rad Laboratories (Richmond, CA, U.S.A.) and Pharmacia (Uppsala, Sweden). Leydig cells purification. Adult male Swiss mice (Janvier, France) were killed by cervical dislocation and testes were immediately excised and decapsulated. Cell suspension of whole testes was prepared as previously reported (11). Ten decapsulated testes were sustained to collagenase digestion in 10 ml medium 199 containing 0.1 % BSA, 3 mg collagenase and 40 ug DNAase I at 37°C in a shaking water bath for 30 min. After dispersion, the cell suspension was washed twice with 0.9 % NaCl, decanted, filtered through nylon gauze (86 Mm) and centrifuged at 150 g for 15 min. The crude intertubular cell preparation was resuspended in 2 ml BSA-medium 199 and layered on the top of a continuous Percoll-gradient as previously described (12). Bands of highly purified Leydig cells were aspirated and 3 vol BSA-medium 199 were added. After centrifugation at 150 g for 15 min, the cell pellets were resuspended in BSA-medium 199 and the total number of purified Leydig cells was determined by counting aliquots in a haemocytometer. Cell viability assessed by trypan blue exclusion was greater than 95 %. The percentage of Leydig cells as determined by histochemical staining for 3/3-hydroxysteroid dehydrogenase was 90-95 %. Perifusion of purified Leydig cells. Theoperifusion method used was as previously described (13). Approximately 10 Leydig cells were mixed with 0,5 g Bio-Gel P2. A 2 ml plastic syringe served as the perifusion column and a peristaltic pump (Bioblock - G 31098) was used to propel the medium through the perifusion chamber at approximately 0.1 mljmin. The perifusate used consisted of medium 199 containing 0.1 % BSA, 7 umolfml L-Glutamine, 350 ug/ml NaHCO , 1 mmol/ ml Hepes, 50 ug/ml gentamycin-sulfate and 4.5 mg/ml glucose. T?Ie columns were placed in a 37°C water bath and the out-flow line was connected to a fraction collector with which eluate samples were When indicated in the results section AVP or obtained at 5 min intervals. The eluates collected were stored at -20°C hCG were added to the medium. Since the rates of testosterone secretion varied until testosterone assay. each experiment (as represented by somewhat from one experiment to another, one panel in the figures) was performed with parallel columns using the same preparation of purified Leydig cells at the same time. Steroid assay. after appropriate

Testosterone dilutions

levels in the eluate from columns were assayed by a specific radioimmunoassay as previously

217

Vol. 146, No. 1, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

described (11) and expressed as ng/1063P-HSD-positive cells. The sensitiThe inter and intra-assay coefficients vity of the assay was 7 pglsample. variation for the assay were < 8 %. The cross-reaction with W-dihydrotestosterone was 36 % but was < 2 % with other steroid hormones.

of

RESULTS Dose-response rone

was

of Leydig achieved

which

time

the

(Fig.

1A).

After

trations testosterone

when

level

ranging

of

As shown

the

end of the

Leydig

cells

were

testosterone

equilibrium

from

0.01

production.

min.

to hCG. A stable

this

presented

lation,

cells

time,

in Fig.lA

(lag

a maximal

experiment

induced

c+c ++ H *+

11

10

period

around

control 0.01 rig/ml hCG 0.1 rig/ml hCG 1 rig/ml hCG

of testoste-

for

about 0.5

6-

in

hCG-stimu-

< 15 min)

to a peak

was obtained

with

1 ngfml

testosterone

levels

value

by 100 hCG. At remai-

l . .

H no incubation H12h incubatior *-+ 18h incubatior

1 o-

6

fraction

increase

acute

0

9-

ng per

at

of hCG at concen-

after

6 h, the

9

60 min,

a dose-dependent

profiles

response

lasting

perifused

a 5 min infusion

The testosterone rise

release

was approximately

to 1 rig/ml,

a rapid

basal

;:l 4

12

Figure

1.

20

28

Effect response

taining

36

of

44

52

hCG and of of perifused

freshly

60

68 Fraction

4 number

preincubation mouse Leydig

isolated

12

period cells.

(A) or preincubated

20

28

on the Parallel

36

44

testosterone columns

con-

mouse Leydig

cells

for 0, 12 and 18 b in hormone free-medium (Bj, were perifused for 6 h. Eluate samples were collected at 5 min intervals. Where indicated by the arrow, the columns were infused for with hCG. The data were typical of 3 separate experiments,

218

52

60

5 min

68

Vol. 146, No. 1, 1987

BIOCHEMICAL

AND BIOPHYSICAL

l.

E T

.

P

.

.

00

-AVP

M

AVP

.

0

(10-5M)

l

.

!2 0

RESEARCH COMMUNICATIONS

0

.

l

0

*

0.5

l 0

5 zLT

0

F 2

-.

4

12

20

28

36

FRACTION

Figure

2.

44

52

60

NUMBER

Effect of AVP pretreatment on basal testosterone secretion. Purified mouse Leydig cell were preincubated for 12 h in the -5. presence or absence of 10 >I AVP. After this incubation period, the columns were washed for 30 min and perifused in parallel for 6 h with hc>rmone-free medium. The data were typical of 3 separate

experiments.

ned,

however,

1 rig/ml

of hCG were

a longer not

than

higher

time

the basal

used,

period

the

and

values.

When concentrations

stimulation

a slow

was maintained

time-dependent

higher

than

at a plateau

decrease

during

was observed

(data

shown).

Effect

of

shows

that

the

compared

response for

at

end of

incubation

Effect

effect

Leydig

cells

that

cells.

Fig,lB

The loss

of the

cell-incu-

examination

cell-viability

hCG.

was markedly

60 and 86 % after

Microscopic

showed

on basal of

profiles

terone

secretion

preincubated

show that over

analysis to

(P < 0.02,

and hCG-stimulated

AVP on basal

simultaneously

cretion

corresponding

Leydig

was about

to

cell-incubation

isolated

18 h respectively.

the

perifused

Statistical

of freshly

experiment

response

to hCG after

peak values

of AVP treatment

containing

higher

the

testosterone

of the

was

cells

unaffected

by

period.

To determine

were

to that

12 h and

on

response

to hCG at the

bation

the

period

testosterone

decreased

the

incubation

with

showed 60 min

testosterone with

that

n = 5 ; paired

testosterone the

start t-test) 219

10 -5M

without

cells

of the

production.

secretion,

medium.

of the

throughout

after

or

hormone-free

pretreatment

controls

testosterone

two

columns

AVP for

12 h ,

The testosterone

se-

by AVP enhanced

perifusion

period

secretion

in

of

perifusion

in

AVP-pretreated

was

the

testos(Fig.

2).

fraction

significantly cells

than

in

Vol. 146, No. 1, 1987

BIOCHEMICAL

AND BIOPHYSICAL

Table

RESEARCH COMMUNICATIONS

1

Purified mzyse Leydig cells were preincubated for 12 h with or without 10 M AVP. After this time, the two separate columns were washed for 30 min and perifused in parallel with hormonefree medium. Each value represents testosterone secretion in the fraction corresponding to 60 nin after the start of perifusion. Values given were from 5 different experiments. TESTOSTERONE

(ng/5

min)

TREATMENT

EXP.

CONTROLS

AVP (10-51Q

1

0.581

0.673

2

0.463

0.582

3

0.593

0.720

4

0.656

0.989

5

0.532

0.616

AVP-treated significantly paired t-test).

controls

(Table

0.573

Zk. 0.038

cells

respectively.

1). and

different

from

The means 2 s.e.m. 0.756 In

f

0.077

contrast,

controls

(P < 0.02

(ng testosterone/5

in

control

the

and

min)

in

pretreatment

;

were

AVP-pretreated of

Leydig

Leydig

cells

by AVP

B 5-

Ca

-AVP

H

AVP

(10-5M)

I?

4-

l-

4

Figure

12

20

3.

28

36

44

52

60

68 FRACTION

4 12 NUMBER

20

28

36

44

52

80

Effect of AVP treatment on the hCG-stimulated testosterone response. Freshly purified mouse Leydig cells were continuously perifuz$d in parallel columns with medium containing or not 10 M AVP (A). Mouse Leydig cefls were preincubated for 12 h in the presence or absence of 10 M AVP. After this incubation period, columns were washed and perifused simultaneously with hormone-free medium (R). Where indicated by the arrow, the columns were infused for 5 min with 1 rig/ml hCG. These data were typical of 3 separate experiments.

220

68

Vol. 146, No. 1, 1987

induced

a marked

compared

to

40 % under -with

BIOCHEMICAL

decrease

the

untreated

control

a longer

of

at the

isolated

peak

effects

Leydig

testosterone

This

cells

of

of

were

reduction

values.

period

and inhibitory

freshly

hCG-stimulated

(Fig.3B).

AVP-pretreatment

stimulatory

RESEARCH COMMUNICATIONS

the

cells

levels

AND BIOPHYSICAL

Similar

the

was approximately results

cells

AVP were,

(data

however,

continuously

production

were not

not

perifused

with

obtained

shown).

Such

observed

when

AVP (Fig.3A).

DISCUSSION The

present

study

testosterone ported

production

by the

dogenic tion

fact

capacity our

show

respectively

on are

oxytocin rat

decrease

the

in

this It

is

this

is

observations those

the

cultured

exposed that

authors

is The

to

(16)

such

due

perifusion

to

by a pure an effect

cells

methodological system

by

has been 221

to

hCG.

AVP alone.

stimulatory Leydig

of

A more were To

our

effect

of

has been

reported

for

oxytocin

(15).

such from

the

rat.

differences used

for

a our

preparation.

a stimulatory

largely

since

levels

observe

species

from

an accumulation

production

or

hypothesis

result

testosterone

derived

AVP and

cell

of

not

the

testosterone

cells

These

in cultured

activities,

of a direct

basal

interstitial

could

basal

concentrations

who did

synthesis

Leydig

production

low

with

exposed

demonstration

that

agreement

cells

effects

both

production

Leydig

the

AVP stimulates

testicular

discrepancy

of

In addi-

production.

showed

that

steroi-

system.

observations) of

sup-

the

show that

17,20_desmolase

however,

first

to note

of other

unknown.

perifusate

strongly

and negative

which

in

influence

affects

positive

data

also

(unpublished

on testosterone

cells

are

is

testosterone

recent

and

pretreatment

interesting

luteal

exerts

on testosterone

is,

This

testosterone

They

effect

the

after

peptide

with

directly

in a perifusion

hCG-stimulated

(14).

observation

knowledge

cells

hormone

and

results

progesterone

increased

Leydig

17a-hydroxylase

own preliminary

testis. peptide

gonadotropin-stimulated

inhibitory

surprising

mouse

AVP can

neurohypophysial

consistent

cells

of

adult

this

basal

inhibit

this

this

that

partly

testicular

that

by the

that

that

indicates

of purified

data

results

clearly

Our kinetic differ

from

effect Whether is

the

human

in this

presently

study

of

the

BIOCHEMICAL

Vol. 146, No. 1, 1987 steroidogenic

regulatory

analogies

with

in static

cificity

could

cells

are

affect

rone

production

testis

point

out

On the

other

incubations. discrepancy.

be that Such

As yet

to

AVP exposure

of

by

steroid

hormones

were

undetectable

stimulatory for

phenomenon

of

proteins

is

In conclusion, the

ficance

of

de-novo

processes our

Leydig

peptides

studies

are

these

peptides

with

other

testicular

of

further

new

steroidogenic

capacity

in the

overall

side-chain in

the

of the

basal

conditions,

AVP occurred

rat

steroi-

The present

C21 results

only

puzzling.

after

A similar

agents

such

as

AVP response

as a func-

AVP-receptors,

regulatory

both

modulators

to elucidate

A likely

the step

also

study

investigations.

AVP affects

as local

cells.

regulatory

of the

locally

testoste-

suggested

in

of

that

required

cholesterol

perifusate.

appearance

needs

act

the

not spe-

The present

Leydig

AVP are

induction

results

cell

neurohypophysial Additional

to

the

the

to

of basal

been

are

mouse Leydig

known

the

since

effects

cells

for

Whether

due

or to other

tively

reported

(19).

time

inhibitory

12 h of Leydig

has been

catecholamines tion

and

in

are

some

a species that

(1.8).

regarding

AVP exposure,

that

increase

by AVP has

we have no information

phenomena hand,

rat

increases

a regulation

affected

pretreatment

in the the

peptide

presents

which

for

the

which

noteworthy

cause

pathway

both

is

agonists

capacity

dogenic

that

It

precise

response

activity. (16).

could

the

in could

cleavage

situation

androgenic

to identify

explanation

model

to LHRH and its

testicular

has failed

This

this

unresponsive

the

(17).

long-term

explain

RESEARCH COMMUNICATIONS

mechanisms

the --in vivo

revealed

AND BIOPHYSICAL

the

function

positively

support

the

of exact

and negafinding

testicular

function.

physiological

of the

that

signi-

testis.

ACKNOWLEDGEMENTS The authors wish to Mrs M. Verger for careful

thank Dr L. Cedard for her preparation of the manuscript.

critical

comments

and

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