Discordant regulation by luteinizing hormone of ornithine decarboxylase activity and testosterone production in isolated rat testicular cells invitro

Vol. 112, No. 2, 1983 April

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

29, 1983

Pages 496-501

DISCORDANT REGULATION BY LUTEINIZING HORMONE OF ORNITHINE DECARBOXYLASE ACTIVITY AND TESTOSTERONE PRODUCTION IN ISOLATED RAT TESTICULAR CELLS -__ IN VITRO J. Medical

Received

Osterman,

E.P.

Murono,

T. Lin

and H.R.

Nankin

Service, Wm. Jennings Bryan Dorn Veterans' Hospital Department of Medicine, University of South Carolina School of Medicine, Columbia, South Carolina 29208

March

17,

and

1983

SUMMARY: Possible functional relationship between luteinizing hormonestimulated ornithine decarboxylase and testosterone production was examined in rat testicular interstitial cells in vitro. Although luteinizing hormone enhanced both ornithine decarboxylase activity and testosterone production at a similar physiological dose range, we found dissociation in the two responses in terms of their temporal aspect and the way they were affected by an irreversible inhibitor of ornithine decarboxylase, alphadifluoromethylornithine, and protein synthesis inhibitor cycloheximide. The results suggest that there appears to be no causal coupling between luteinizing hormone-stimulated enzyme activity and testicular steroidogenesis.

Cellular are

associated

synthetic

less

with

enzymes

coupling is

responses

to various

marked

well

thine

decarboxylase

amine

biosynthesis

examine

also

possible

decarboxylase

of the activity

levels

with

agents

of polyamine

(l-3).

However,

specific

cellular

the

biofunctional

response(s)

understood. demonstrated

by LH of testicular

of LH that

and growth-promoting

accumulation

polyamine

We have recently tion

enhancement

and polyamine

of increased

hormones

interstitial

activity

augment

and dose-dependent and purified

the rate-limiting

stimulation

testicular

functional

cell

in vitro, -___ This

(4).

activity

a specific

occurs

steroidogenesis.

relationship

between

and testosterone

production

Leydig

stimulacell

orni-

enzyme of poly-

with

physiological The present

LH-stimulated

doses studies ornithine

by the same cells

in

vitro.

Abbreviations

Used:

DFMO; Alpha-difluoromethylornithine;

0006-291X/83 $1.50 Copyright 0 I983 by Academic Press, Inc. All rights of reproduction in any form reserved.

496

LH; Luteinizing

Hormone

Vol. 112, No. 2, 1983

MATERIAI-S --

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

AND METHODS

Chemicals: Ovine LH (NIH-LH-S23) was obtained from the Hormone OistribuEmce (NIAMMD, NIH, Bethesda, Maryland). Collagenase (Type I), 3-isobutyl-l-methyl-xanthine and cycloheximide were purchased from Sigma Chemical Company, St. Louis, Missouri. L-[l-14C] Ornithine monohydrochloride (sp. radioactivity 58.9 mCi/mmol) was obtained from New England Nuclear Corporation, Boston, Massachussets. Culture Medium 199 with Hank's salts and 25 mM Hepes [4-(2-hydroxyethyl)-l-piperazine-ethanesulphonic acid1 buffer (pH 7.4) was from Gibco, Grand Island, New York. DFMO was generously provided by Dr. D.J. Wilkins, Centre dRecherche Merrell International, Strasbourg Cedex, France. Animals and Tissue: Male Sprague-Dawley rats (60-120 days old) were purchased from Zivic-Miller Laboratories, Inc., Allison Park, Pennsylvania. Testes were quickly removed from decapitated rats, decapsulated and kept on ice. Testicular interstitial cells were prepared by collagenase digestion as previously described (5). The incubation with collagenase was carried out for 15 min. Culture Conditions: Interstitial cells (1.5-2.5 x lo7 viable cells, determined by Trypan-Blue exclusion) were incubated in 10 ml of Medium 199 containing 1.0 mg/ml bovine serum albumin and 25 mM Hepes buffer, pH 7.4 (culture medium) for 1-4 h at 340C in an atmosphere of 02/CO2 (95:5, v:v). LH, in concentrations as indicated, 0.1 mM isobutyl-methylxanthine (dissolved in dimethyl sulfoxide and added in the total volume of 0.5 Pi/ml culture rrledium), cycloheximide and DFMO were added at the beginning of the incubation. Control culture received a corresponding volume of the solvent. Following incubation cells were recovered by centrifugation (at 600 x q), washed twice in homogenizing medium containing 0.25 M sucrose and then homogenized in 2.5 ml of ice-cold 25 mM Tris/HCl buffer, pH 7.4, containing The homogenates were centrifuged for C.l mM EDTA and 5.0 mM dithiothreitol. 30 min at 20,000 x g and supernatants were used for the assay of ornithine cecarboxylase activity and protein concentration. Enzyme Assay: Ornithine decarboxylase activity was determined by neasuring in duplicate the liberation of 14C02 from t-[1-14C] ornithine under conditions as previously described (6). The enzymatic activity was expressed as pmol of CO2 released/mg cytosolic protein per 30 min. Cytos#olic protein concentration was determined by the method of Lowry et al (7) as modified by Munro and Fleck (8), with bovine serum albumin as the standard. Testosterone Radioimmunoassay: Testosterone was immunoassay on ether-extracted aliquots of incubation chromatography, following procedures which have been Results are expressed as ng/106 cells per time (5). 4 h). Results were analyzed by Student's t test.

measured by radiomedium without prior described previously of incubation (usually

RESULTS -___ We have recently by LH of rat (4).

testicular

The minimally

stimulation the respective

reported interstitial effective

of the enzyme control

a specific cell

and dose-dependent ornithine

decarboxylase

dose of LH was 10 pg/ml activity,

in several

which independent 497

varied

stimulation in vitro -___

and the maximal

between

experiments,

40-100%

above

was achieved

BIOCHEMICAL

Vol. 112, No. 2, 1983

AND BIOPHYSICAL

I

TABLE

__-

LH-Stimulated ---

Treatment

RESEARCH COMMUNICATIONS

Testosterone

Testosterone

Production

(ng/lOF

cells

per

4 h)*

P **

.-___Control LH 1.0 10.0 100.0 1.0 100.0

3.14 3.23 6.41 11.83 11.89 12.36

pg/ml pg/ml pg/ml rig/ml rig/ml

*Results are ** vs control

means

-+ S.E.M.

LH dose of 100 pg/ml

with

by interstitial although

cells

of

+ + i T T +-

0.03 0.06 0.14 1.05 0.15 0.20

3 incubations

at 4 h of incubation

(4).

as shown

in Table

I followed

of this

response

(about

the magnitude

~C.001
Testosterone a similar

4-fold

production

dose response,

stimulation)

was much

of ornithine

decar-

higher. To examine boxylase

and testosterone

irreversible marked

inhibitor

depletion

in Table thine bition

the relationship

II,

production

DFMO caused

(lo-12%)

decarboxylase,

polyamines

activity of testosterone

of

but

0 100

100 100 100

an enzyme-activated DFMO, which systems

inhibition

produced

(9).

As shown

no statistically

orni-

significant

inhi-

production. II

Ornithine Activity per DFMO

causes

of LH-stimulated

DFMO on LH-stimulated Ornithine Decarboxylase and Testosterone Production

Treatment

LH (w/ml)

in several

a dose-dependent

TABLE Effect

the activity

, we employed

of ornithine

of cellular

decarboxylase

between

Decarboxylase (pmol/mg protein 30 min)

Activity

Testosterone (ng/106 cells

per

4 h)

(mM)

0 0 0.1 1.0 10.0

37.86 53.81 30.58 22.97 22.18

Results are means + S.E.M. of the beginning of 4-h incubation. < 0.02 vs control :*p p < 0.005 vs LH only *** p < 0.001 vs control

+ T T T 5

2.67 2.12* l-82** 2.63** 1.36**

3 incubations.

498

1.22 4.17 4.21 3.74 3.69 LH and

+ + T + z

DFMO were

0.02 0.15*** 0.13*** 0.15*** O.lO*** added

in

BIOCHEMICAL

Vol. 112, No. 2, 1983

Since

gonadotropin-stimulated

continuous

synthesis

inhibited

by protein

has of interest boxylase

ornithine

produced

molecules

of this

in Leydig

and further

loo-fold

no statistically

was blocked increase

significant

previous

inhibited

III).

(Table

additional

LH-

In contrast,

by the

in the

it decar-

with

and completely

activity

(10,ll)

on ornithine

In agreement

cells

upon

and can be completely

inhibitor

dose-dependently

decarboxylase

depends

such as cycloheximide

production.

synthesis

of cycloheximide

jnhibitor

the effect

RESEARCH COMMUNICATIONS

steroidogenesis

inhibitors

cycloheximide

testosterone

LH-stimulated cases

synthesis

and testosterone

(10,ll)

stimulated

testicular

of new RNA and protein

to compare

activity

reports

AND BIOPHYSICAL

two lowest

dose of the inhibition

of

the enzyme activity. Furthermore, crnithine the

analysis

decarboxylase

enzyme activity

bation!,

compared

of temporal activity

actually

linearly

between

and testosterone

decreased

to freshly

duction continued

relationship

during

isolated over

the

cells

the first (4),

entire

production

while

LH-stimulated showed

two hours

that of incu-

testosterone

4 h of incubation

pro-

(results

not

!;hown). [IISCUSSION --~ Pituitary c:hief

gonadotropins,

regulators

follicle-stimulating

of testicular

function, TABLE

Effect of (ng/lOh cells

Cycloheximide per 4 h) (pmol/mg

hormone

have been shown

on Testosterone Production and Ornithine Decarboxylase protein per 30 min)*

Treatment

0 100 100 100 100 100 *Results added

Cycloheximide

to stimulate

two tes-

III

Activity

-

LH (Wml)

and LH, the

Testosterone

Ornlthlne Decarboxvlase

(ug/ml)

0 0 0.05 0.25 2.5 25.0 are means + 5.t.M. in the beginning of

2.22 + 0.02 11.42 i 0.22 8.78 T 0.29 4.83 + 0.12 2.24 + 0.04 1.64 + 0.05 of 3 incuoations. LH and 4 h incubation

a p < 0.02 vs control b p < 0.01 vs LH only 499

32.84 + 46.89 + 36.77 + 31.85 i 27.95 i 26.28 + cyclohexlmide

1.54 1.70a 0.51b 1.74b l.lOb 3.20b were

Vol. 112, No. 2, 1983

BIOCHEMICAL

titular

ornithine

decarboxylase

rodents

(12-14).

More recently,

vitro from

effect adult

of LH on this rats

biosynthesis

(4).

results

of the

following

enzyme

regulation

of these

there

two responses. decarboxylase

but produced

no statistically

reported

by Danzin

et al

in porcine

stimulated

progesterone

differences

are

of ornithine

ovarian

level

degree

depletion

A recent

in vivo -__

DFMO administration

was employed

was not

required

Cycloheximide 95% inhibition In view higher

degree

it

was surprising

of inhibition granulosa

cells

pletely

inhibited

both

LH-stimulated

production

(16).

ornithine

in isolated that

in vitro -__

It is possible 500

Whether

a remains

intratesticular

to cause

it

not

about

cell

produce

activity.

in our

activity cell

(18). a In

(10 pg/ml)

decarboxylase that

testi-

(17).

Leydig

, cycloheximide

ornithine

degree,

decarboxylase

testis

decarboxylase

inhibition

preparation

production

did

LH-

tissue the

(15).

in which

was reported

to find

ovarian

affecting

though

in our cell

androgen

of ornithine

that

administra-

those

depleted

that

testicular

synthesis

for

to that

of the enzyme

without

in rabbits

suggested

porcine

progesterone

occurred study

in vivo -__

was of a moderate

in the dose of 25 ug/ml of protein

of this,

for

testis

to be markedly

of polyamine

to be determined.

activity

It is of note

in the rat was found

--in vitro

synthesis.

was similar

degree

The reason(s)

(16).

inhibitor

of testosterone

following

cells

coupling

of the enzyme activity

a much higher

understood.

decarboxylase

testis

granulosa

synthesis

not well

putrescine

similar

DFMO produced

at a similar

irreversible

impairment

rat

ornithine

functional

by DFMO we observed

in the

The

cells

59% inhibition

inhibition

(15)

inhibitor.

inhibition

about

such

understood.

to be no apparent

significant

of the enzyme

tion of this

cular

caused

polyamine

LH stimulates

DFMO, an enzyme-activated

of ornithine

The magnitude

poorly

in

cells

functions

in interstitial

appears

direct

interstitial

testicular

although

production

time

of enhanced

remains that

to immature

the first

testicular

significance

suggest

decarboxylase and testosterone dose range,

administration

of differentiated

study

RESEARCH COMMUNICATIONS

for

in isolated

and steroidogenesis present

physiological

in vivo -__

we demonstrated

The functional

in the

as spermatogenesis

AND BIOPHYSICAL

comand

preparation

BIOCHEMICAL

Vol. 112, No. 2, 1983

cycloheximide

was affecting

both

AND BIOPHYSICAL

ornithine

RESEARCH COMMUNICATIONS

decarboxylase

synthesis

and

degradation. In conclusion, tie no causal

in rat

coupling

and testosterone

testicular

between

interstitial

LH-stimulated

ornithine

cells

there

appears

decarboxylase

to

activity

production.

ACKNOWLEDGEMENTS -__ This work was supported by the Veterans Administration and the National Institutes of Health/National Institute of Aging Grant 1 ROl AG 01217-03-REB. We thank E. Patterson and D. Barnett for excellent technical assistance and Mrs. A. Martin for secretarial assistance.

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