An invitro action of androgens on protein synthesis by epididymal tubules maintained in organ culture

An invitro action of androgens on protein synthesis by epididymal tubules maintained in organ culture

Vol. 52, No. 4, 1973 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS AN --IN VITRO ACTION OF ANDROGENS ON PROTEIN SYNTHESIS BY EPIDIDYMAL TUB...

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Vol. 52, No. 4, 1973

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

AN --IN VITRO ACTION OF ANDROGENS ON PROTEIN SYNTHESIS BY EPIDIDYMAL

TUBULES MAINTAINED Jorge

A. Blaquierl

The Population The Rockefeller New York City, 10021

Received

April

25,

IN ORGAN CULTURE

Council University New York

1973 SUMMARY

The incorporation of 14 C amino acids into protein by rat epididymal tubules maintained in organ culture was studied. It was found that the incorporation of labelled amino acids increased with the duration of the culture. The addition of dihydrotestosterone 10e5M to the media during 3 days' culture produced a 130% stimulation on the amino acid incorporation. When the tissue was kept in culture for 7 days, both testosterone and dihydrotestosterone at LO-'M increased the amino acid incorporation (150% and 118% respectively). This --in vitro effect of androgens was blocked by the presence of Actinomycin D (20 pg/ml) in the medium. Neither estradiol 178 nor progesterone or corticosterone induced any changes in amino acid incorporation. Although

the epididymis

dependent

organ,

by these

hormones.

Recently, cytoplasm

control

and nuclei

of the organ, (7)

has also

Presently, protein

is know about

the presence

(l-4)

function

little

has been

with

we wish

synthetic

the

of specific (5-6)

been

ligated supply females 1

with

5-O silk

of either for

On leave

testes

3 weeks. of absence

control

respect

many years

of its

receptors

for

as an androgen-

metabolism

androgens

Evidence

to sperm maturation,

and function

in

the epididymal

suggesting

is

under

that

the

androgenic

presented. to report

function

efferent

for

was demonstrated.

on the --in vitro

of this

tissue

MATERIALS The right

recognized

ducts

of adult

sutures, or caput

from

the National

in

of androgens organ

on the

culture,

AND METHODS (300-350

g body weight)

avoiding

epididymis.

procedure

Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

maintained

carefully

This

effect

yielded

1177

any interference

The animals epididymis

Research

Holtzman

Council,

were free

with

rats

the blood

caged with of spermatozoa

Argentina

were

normal while

BIOCHEMICAL

Vol. 52, No. 4, 1973

the

contralateral

testis

preservation. the

capsule

After

technique

treatment

and placed

over

containing

10% fetal

fungizone

of agar calf

aseptically

for

1 hour

in organ

to each dish

adequate

and cultured (8).

in 0.05%

was dissected,

and penicillin,

was assayed salt

Briefly, Pronase.

straightened

culture

dishes.

streptomycin

(100

and the culture

amino

acids

95% air

and stopped

2 mg/ml

Casaminoacids

by incubating

solution

acid

was carried

with

mixture,

out

by the addition (caseine

pieces

supplemented

(14 C amino

The incubation

Mass.).

serum

for

and Tichenor

digested tubule

necessary

out MEM medium W/ml)

incubated

and

at 31°C

of 5% CO2 in air.

balanced

of "C

removed

(2%, Difco)

was added

synthesis

of Earle's

tissue

RESEARCH COMMUNICATIONS

balance

by Orgebin-Crist

and the

a film

(5 pg/ml)

Protein

were

the epididymal

an atmosphere

$/ml

described

was opened

this

under

the hormonal

The empty epididymides

following the

provided

AND BIOPHYSICAL

of

in 1 ml

1 mM methionine New England

at 31°C under

Difco)

and 0.01

Nuclear,

an atmosphere

of 0.5 ml of Earle's

hydrolysate,

the tubule

solution

Boston, of 5% CO2:

containing

and by freezing

the

incubation

blotted

on

mixture. The incubated

tissue

filter

paper

were

removed

cold

10% TCA was added.

filter, then

and homogenized for

washed dried

added

was removed

protein

with

in vials

to each

determination

were

prepared

to the medium Actinomycin 20 pg/ml method Student's

as

test

counted.

stock

into

Protein acid

testosterone

solution

1 ml of

Whatman GF/A glass

of ethanol:ether

(1:l)

scintillation synthesis

insoluble

fluid

(10m3M in 25% ethanol

other

were

was expressed

material/mg

(T) and all

and

D (Sigma

Chemical

immediately

et al.

(9) using

was applied

Co.)

prior

was prepared

to use,

crystalline

to determine

Protein

steroids

in saline)

bovine significance.

1178

at a concentration was measured serum albumin

as

of protein. used and added

such.

of medium of Lowry

(DHT),

as 100X

a mixture

homogenate

over

Ten ml of toluene-based

incorporated

Dihydrotestosterone

Two 100 ~1 aliquots

water.

was collected

5% TCA and with

and the samples acids

medium,

and to the remaining

The precipitate

excess

the incubation

1 ml of distilled

at 1OO'C.

vial

cpm of 14 C amino

in

from

of by the

as standard.

BIOCHEMICAL

Vol. 52, No. 4, 1973

50 45

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

55

Controls Different I” culture

3 days I” culture T and DHT IO-J M

t,mes l

7days

50

/ 40 ; ‘0

35-

L

30-

/ *A

? N\ bx E g

5doys

25/i ml5-

IncLlbotlon

time

(mln) 60

30

lncubatlon

Figure 1. 'The l4 C tubules maintained were incubated for in Earle's balanced &i/ml (14C) amino Figure 2. Effect at lo-5M in the tained in organ D (20 pg/ml) was

120

time

(mln)

amino acid incorporation into protein by rat epididymal in organ culture for different periods. The tissues O-120 min at 31°C under an atmosphere of 5% CO :9X air salt solution supplemented with 1 mM methionr *a e and 0.01 acids.

of the medium culture added

presence of dihydrotestosterone (DHT) and testosterone on amino acid incorporation by epididymal tubules mainfor 3 days (Mean f S.E.). Where indicated, Actinomycin to the culture mediu-

RESULTS The protein organ

culture

increased

day in culture

a three-fold

culture amino

resulted acid

testosterone poration,

acid but

increase

The presence

activity with

of C14 amino

poration first

synthetic

into this

after

the epididymal

duration acid

almost in organ

of dihydrotestosterone

incorporated

large

into

(10W5M) also the

material doubled

0.025)

an augmentation was less

than

1179

that

incorthe

1).

in the amount In a parallel the C14 amino

effected

after

3 days and showed

the medium during

in

in

little

was detected

(Figure

increase

fraction.

maintained Very

after

culture

10e5 M in

( the protein

produced

increase

(P

tubules

of the culture.

insoluble

activity 7 days

in a very

although

the

of

a

3-day

of Cl4 experiment, acid

by DHT (Figure

incor2).

BIOCHEMICAL

Vol. 52, No. 4, 1973

T and

I20

AND BIOPHYSICAL

DHT

RESEARCH COMMUNICATIONS

&‘M I T lO-5 M

100 I

E” 2 b-

DHT

70. 60-

60

30

120

lncubatlon

When the

tissue

the ability

duced

protein

stimulation (Figure

under

the influence

values

(P (0.025),

significantly tration

of 10-/M,

tissue

The amount

that

not

and then

both

acid

under were

greater

added

as clear

amino

incorporated

hormone

acids protein

than

the

did

to the medium obtained

pro-

into

stimulation

as those

tested

androgens

of radioactive

T was significantly

When androgens were

7 days

was found

of amino

3).

the results

for

incorporation

the values

control

not

differ

at a concenwith

the use

of hormones.

of Actinomycin

and DHT (10e5M),

steroids

the

although

of the androgen-stimulating other

(m(n)

hormones it

of DHT and with

concentrations

The addition

with

protein,

in

3).

(Figure

of higher

the

was cultured

to synthesize

a sharp

into

lime

Effect of the presence of dihydrotestosterone (DHT) and testosterone M in the medium on amino acid incorporation by epididymal tubules mainin organ culture for 7 days (Mean ? S.E.),

==+-loat tained

for

lO-5 M

D (20 pg/ml)

incubated effect

such as estradiol

for (Figure

178,

to the culture

3 days, 2).

progesterone

1180

resulted As control

medium

containing

in a complete experiments,

and corticosterone

block several (10s5M)

were

BIOCHEMICAL

Vol. 52, No. 4, 1973

used

in place

of

of the steroids into

the androgens, induced

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and the

significant

tissue

was cultured

stimulation

of amino

for acid

3 days.

None

incorporation

protein. DISCUSSION The results

poration

of the present

of radioactive

dymis

is

control

also

rather

rises

Preliminary

of

lapses,

organ

progressively

the castrated in

tration

effect

endogenous

same line tissue

indicating 44 rig/g

the as early

that of fresh

acids

incor-

the epidi-

into

in culture. function

This after

the

epididymal

this

view

resembling

respect,

tubule.

with

is

regard

for

the that

explain 7 days necessary

of the physiological the content tissue.

and Vreeburg fall

authors

to

explain

starting

to obtain range.

a water

1181

content

concen-

measurable

for

organ effect

levels

several synthesis

culture.

The

of androgens

cultured

a clear-cut

Nevertheless,

shown

of protein

the

to that

have

the half-life

cultured

the more dramatic as compared

in

why the hormonal

stimulation

of DHT in the epididymis Assuming

below

has been

hormonal after

fell

found

tissular

that

in

and the

(10)

in the

reported

could

tissue

as time

the condition

an abrupt

in turn,

that,

or metabolized

and the amounts

after

would

Aafjes

These

fact

one may suppose

a state

as 24 hours

of androgens

seem to be out

of amino of time

to confirm

is utilized

This,

to detect

in culture

The dose

of

normal

androgen

castration.

of reasoning

kept

tend

there

castration.

can be observed

material

the unconvoluted

lacking,

into

In this

notwithstanding

days,

of the

medium.

incorporation

to obtain

are

epididymis,

(easier

culture

of the tissue's

DHT was 4-5 hours

after is

data

enters

of DHT after

27 hours

insoluble

to the length

observations

animal.

epididymal

a stimulation

the epithelium.

the

the rat

the basal

employed

pertinent

culture

acid

to the

the recovery

histological

Although

that,

that

treatment

the structure

into

proportionally

reflect

strong

demonstrate

acids

of androgens

observed

tissue

may probably

for

amino

by the addition It

study

for

effect there

3 days.

(10m5M) is a report

of the normal of 80% for

only

on

rat

the normal

is

may (10) about organ

BIOCHEMICAL

Vol. 52, No. 4, 1973

(Blaquier,

unpublished

the order lower the

concentration

not It

statistically

requirement possibility

that

the uptake

ration

of

androgen

prior

added

x 10e3M)

androgens

by prostate

since

might

explants

erratic

account

and

for

in which

the

epithelium,

found

by approxi-

authors

showed testosterone

when serum

(5%) was

stimulation

of protein

(12)

protein

animal

by explanted

insulin

when (3.5

effect the

this

x 10S6M)

response

testosterone

present

of

of DHT and T upon

the epididymis

not be reproduced

effect

the tissue

of the action

of DHT by the presence

1182

to must

4 and 2 days of testosterone Insulin

of protein.

Buresova

into

24 hours

levator in a medium

concentration on protein

(1 mg/ml, and RNA synthesis.

seems to be specific

by estradiol

incorpo-

in the medium.

for

At higher

has

in response

( 14 C) leucine

was pre-incubated

testosterone.

in the

the addition

on the synthesis

an inhibitory

could

this

treatment

incorporation

tissue

an increase

with

was also

by androgens

mouse prostate

to obtain

androgen

stimulatory that

demonstrated

pre-treated

prior

synthesis

178,

for

progesterone

or corticosterone. The blockage

of

they

was reduced

of 2 x 10m5M (5 ug/ml)

calf

In support

of the prostate

effect

exhibited

it

in

10% fetal

a marked

unless

testosterone

The action

to proteins

(11)

Lostroh

reported

1 pig/ml

contained

and Franklin

Nevertheless,

a strong

increased

medium

by Lasnitzki

Without

ani

the culture

These

a castrate

(13)

3.3

were

to the medium.

--in vitro.

and Gutmann

containing

DHT and T at a an increase

the values

an effect.

into

was ineffective

protein

toward

in

medium.

leucine

from

se showed

with

to obtain

the --in vitro

to sacrifice.

--in vitro per

for

(14C)

trend

of DHT is

serum was added

and confusing.

be obtained

that

at a concentration culture

Evidence scarce

was detected,

performed

concentrations

the work

to obtain

in the

been

acids

of the hormones

10% calf

had to be added present

a definite

of 3H-testosterone

in order

concentration

were

Although

in mind

steroid

is

40% when

that,

experiments

C amino

the binding

of high

this

tissue

significant.

be kept

serum and thus,

mately

14

of

should

Similar

(10e7M).

incorporation

were

the calculated

data),

of 2 x 10e7M.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of actinomycin

D in

BIOCHEMICAL

Vol. 52, No. 4, 1973

the culture

medium

the synthesis

is

suggestive

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

effect

of androgens

is

induced

through

of RNA (14).

ACKNOWLEDGEMENTS The authc,r

wishes

to thank

Debora

Breger

for

her

skillful

technicaL

assistance. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Blaquier, J.A., Biochem. Biophys. Res. Cormnun. 45: 1076, 1971. Ritzen, E.M., S.N. Nayfeh, F.S. French and M.C.lobbins, Endocrinology 89: 143, 1971. Hansson, U., 0. Djoseland, E. Reusch and S. Aasuik, Acta Endocr. (Kbh.) 71: 614, 1972. &nzo, B.J., M.C. Orgebin-Crist and D.O. Toft, Endocrinology 92: 310, 1973. Calandra, R.S. and J.A. Blaquier, Acta physiol. latinoam. 22: 116, 1972. Tindall, D.J., F.S. French and S.N. Nayfeh, Biochem. BiophG. Res. Conxnun. 49: 1391, 1972. M.S. Cameo and M.H. Burgos, Endocrinology 90: 839, 1972. caquie,r, J.A., Orgebin-Crist, M.C. and P.L. Tichenor, Nature (London) 2397227, 1972. Lowry, 0.) M. Rosebrough, A. Lewis-Farr and R. Randall,T Biol. Chem. 193: 265, 1951. Aafjes, J.H. and J. Vreeburg, J. Endocr. 53; 85, 1972. Lasnitzki, I. and H. Franklin, J. Endocr. 54; 333, 1972. Lostroh, A.J., Proc. Natl. Acad. Sci. (USA) 60: 1312, 1968. Buresova, M. and E. Gutmann, J. Endocr. 50: 643, 1971. Liao, S., R. Barton and A. Lin, Proc. Naa. Acad. Sci. (USA) -55: 1593, 1966,

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