Endocrinol. Japon. 1979, 26 (4), 423-429

Prolonged Stimulation of Adenylate Cyclase Activity and Testosterone Production by Cholera Enterotoxin with Suppression of Gonadotropin Release in Rats KANJI

SATO

AND NAKAAKI OHSAWA

The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113

Synopsis Endocrine in

effects

normal

CET

and

in

the

increased to

bilateral

control

after1-3days; levels

decreased

in of

physectomized

after6hr,

the

level

control

cyclase

in

stimulates

the

increases results days, testicular

in

the

axis

level

weight

and was

adenylate

adenylate

cyclase

of

times

elevated

and of of

serum

the

the LH

of

serum

May16,

1978.

testosterone

in

the

the in

the

was and

cells. of

hypo-

remarkably returned in

CET

testis. prolongedly

Leydig enzyme

cells

and

stimulation for

mechanism

to

adenylate

CET-injected of

concentration

undetectable

Both

increase

including

prolonged

feedback

range

testes

least3days

cells the

returned

germinal

testis

injection

testicular that

of

bilateral

with

enhanced

negative levels

at

and

undetectable

theCET-injected

loss

the

of

after3days.

CET-injected for

of5ƒÊg remarkably

days

the

when

accordance

suggest

It is well known that cholera enterotoxin (CET), an exotoxin of Vibrio cholerae, stimulates ubiquitously adenylate cyclase activity of the mammalian cells studied so far after having bound to its receptor in the plasma membrane, monosialoganglioside, GM1 (Kiefer and Kantor. 1976). CET is, therefore, capable of mimicking various hormonal effects in endocrine and non-endocrine tissues by stimulating their adenylate cyclase/cyclic AMP system, and ACTH-, TSH-, and MSH-like effects have been reported (Donta et al., 1973; Wolff et al., 1973; Mashiter et al., Received

a

intratesticular

activity

elevation stimulation

decrease

activity

in

marked in

remarkably

that

production,

sustained the

content indicate

least3

injection, by

good

at

decreased

the

investigated

concentration

for

significantly

relatively

were injection

decreased

intratesticularly

four In

data

level

accompanied

cyclase

testosterone

to

was

gonads

testosterone

elevated LH

also

injected

male

intratesticular

serum

after

remained

vivo

rats,

significantly

FSH

CET

testosterone

causing

normal

Serum

in7days.

activity,

These

of

on

After

increased28days

rats,

stimulated

(CET)

rats.

in7days.

serum

When5ƒÊg

enterotoxin

remained level

gonadotropin

cholera

testes

after24hr,

the

testis

of

hypophysectomized

at

least3

ofhypophysealrange.

1973; O'Keefe and Cuatrecasas, 1974). Most of these studies were performed, how ever, in in vitro system, in which it is, difficult to observe a prolonged biological effect of CET (Pierce et al., 1971). Previously we reported that CET stimu lated adenylate cyclase activity of rat testis slices with a lag time of about30 min, caused an increase in cyclic AMP accumulation and stimulated testosterone synthesis from cholesterol in vitro, and suggested that these gonadotropic effects were exerted directly on Leydig cells (Sato et al., 1975; Sato and Ohsawa, 1977). This assumption was recently confirmed by Cooke et al.,(1977) using purified Leydig

424

SATO

AND

cells of rat testes. The present studies were undertaken in vivo to investigate the endocrine effects of CET on the rat testis by intratesticular injections.

Endocrinol. August1979

OHSAWA

Japon.

assay (Nieschlag et al., 1972). The minimum sensitivity of the method was40pg of testosterone. Recovery following extraction and thin layer chromatography

was

on the order

of60%.

Experiment2 Nine

Materials

and

to10-week-old

250g

Methods

of

were CET

(5ƒÊg)

Normal

and

tama, at

hypophysectomized

strain

were

Japan)

and

about20•Ž

while

The

was an

inserted

for10sec.

was

Japan

fed

a

intra-

testis

with

of

provided

testis

until

assayed.

Dr.

and

stained was

LH

and

kit

Ohtomo

assay.

1974).

was

with

hematoxylin-eosin.

fixed

by

were

were

samples minimum

for

LH

at-20•Ž and

Bouin's

soluSerum

radioimmunoassay. by

radioimmuno-

NIAMDD

all

10ng/ml

were

removed in

measured the

In

FSH

stored

were

by

for

3,

group

then

them

FSH

40ng/ml

made injected

After1,

each

were

Program.

The

rats.

serum

measured

provided

variation,

control

was

between8and10A.M.. and

of

injection was

saline

from

Testes

testosterone

Hormone

Institute,

of

decapitation

collected

weighing200-

saline

of

5rats

Some

assay

massaged

Research al.,

was

of

volume

testes

by

Blood

rats

intratesticular

in100ƒÊl

same

bilateral

Serum

slowly

single

and28days,

tion A

the

gently

from

et

ether.

was

was

the 14,

weighed.

methanol.

middle

The

sacrificed on

All

(60or100ƒÊl)

(Ohtomo

7,

maintained

libitum.

Chemo-Sero-Therapeutic

Kumamoto,

into

between8and10

the

the

Wistar(Sai-

and

anesthetized

into

CET

the

the

room

with80%

volume

Thereafter,

a

made

were

of

Dohanken

studies ad

cleansed

appropriate

injected.

at

were

was

in

chow

animals

scrotum

needle

kept

rat

injections

A. M.

from

these

laboratory

testicular

and

were

throughout

standard

rats

purchased

A

dissolved

bilaterally. Imamichi

normal

used.

order

Rat

to

measured

sensitivity

the

Rat

(NIAMDD

Rat

about200g

were

interassay

within of

(NIAMDD

Pituitary

avoid

FSH LH

a single

method

was

RP-1)

and

RP-1).

Experiment1 Five ƒÊg injected

of into

rats.

The

the

CET

the same

right

of

was

weight,

After

immediately

aorta

and

rated

and

stored

testosterone cording

the

Frozen

testes

were

Polytron

PT-10

homogenate

of

ethyl

acetate

by

centrifuged

acetate

was

extraction ethyl of

was

water

residue

and

gel,

20)(Dufau separated eluted N2gas. and

once

evaporated

applied

to

et from with4ml Residues

appropriate

al.,

1972).

The

methanol, were

aliquots

The

combined with3ml The

chromatography

and developed chloroform: ether testosterone

which

cut

was

in2ml for

out

dried of

and under

methanol

radioimmuno-

washed

of

to

1977).

Enzyme

rected

for

radioactivity

per

of

used

the

activity

the as

protein

at6for30sec.

in

absence picomoles per

for5min centrifuged

at

min.

pellets buffer

the for

method

chromatographic

expressed mg

in

same

solution buffer

adenylate of

modifications

in

set

homosolution

The1,000-10,000g

was

slight

and

Tris-HCl

protein

with

experiment1.

further

resuspended

according

im-

measurement

at1,000g

was

at4•Ž.

then

immediately

sucrose dial

the the

the

centrifuged

CET

and7days,

for

in

testis of

into

decapsulated

with25mM

and

(1974)

used

supernatant

for20min

assay

was

and

the

was

The

5ƒÊg

ice-cold0.25M

PT-10with

50-80ƒÊg

was twice (80:

fraction

was

used

ethyl

N2gas.

layer

dissolved were

the

twice

dihydrotestosterone of

and

of

the

injected

testis

was

pituitary

and

72hr,

described testis

homogenate

were

24, A

as

Polytron

was

operation of

operation,

saline

nitrogen

the

absence weight

the

After6,

liquid

of

the

body

after of

in5ml

10,000g

in

days

contralateral

(pH7.4)

tube.

and

under

thin

dial

The

washed

Merck) system of

the

for5min,

another

more

was

by

decrease

testosterone

The

by

removed.

in

at4•Ž.

with8ml

for10min. to

then

20•~20cm, in the

Nuclear)

with

were

The

by of

extracted

mixing

transferred

fraction

was

(silica for50min

Vortex

repeated

acetate

ice-cold

autopsy

testes.

hypophysectomized

Completeness

in60ƒÊl

genized

homogenized

for30sec was

at3,000rpm

phase

al.(1972).

England

then

(Kinematica)

The

then

New

dissolved

of

ac-

of

at the

mersed

Serum

age.

Seven

testes

sepa-

and2,000cpm

and

at8.

was

in3ml

assessed

bilateral

abdominal

et

thawed

set

blood

assayed.

was

of

weight. in

testes,

the

Nieschlag

recovery

were

radioimmunoassay

(40Ci/mmol,

monitoring

the

until

methanol

[H3]testosterone

testes

Serum

by of

containing15%

for

of

weighing

at9weeks

remnants,

immersed

for3hr.

measured method

The

from

clot

the

pentobarbital

immediately removal

Experiment3 Rats

in

After24hr,

i. p.).

at-20•Ž

was to

saline

to

was

injected

sodium

withdrawn

allowed

was

rats.

and

nitrogen.

saline

normal

saline

with

body

weighed

liquid

of

anesthetized

(50mg/100g

of

of54-month-old

of5control

were

removed,

in100ƒÊl

testis

amount

testis

animals

dissolved

right

(Sato duplicate

and cyclase

Salomon and

et

samples,

cor-

recovery

and

of

homogenate,

of

testis cyclic

AMP

al.

Ohsawa,

for

formed

the

Vol.26,

No,4

CHOLERA

TOXIN

Table1.•@

Five ƒÊg rats.

of

CET

dissolved

as

After24hr, described

NS:

in

not

();

the

Testosterone

in100ƒÊl

testosterone

saline

in

The

ADENYLATE levels

of

content

Methods.

AND

the

results

or

testis

were

given

in

serum

saline and

and

alone

was

concentration as

425

CYCLASE testes.

injected in

into

the

the

serum

right

were

testis

of

determined

means•}SE.

significant,*p

Prolonged stimulation of adenylate cyclase activity and testosterone production by cholera enterotoxin with suppression of gonadotropin release in rats.

Endocrinol. Japon. 1979, 26 (4), 423-429 Prolonged Stimulation of Adenylate Cyclase Activity and Testosterone Production by Cholera Enterotoxin with...
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