Regulation of Sertoli cell cyclic adenosine 3′:5′ monophosphate phosphodiesterase activity by follicle stimulating hormone and dibutyryl cyclic AMP

Vol. 98, No. 4,198l February

BIOCHEMICAL

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

27, 1981

Pages

REGULATION

3':s'

OF

MONOPHOSPHATE

M. lstituto

HORMONE

Conti,

di

R.

CELL

AND

Geremia,

Istologia

ed

Via

December

SERTOLI

CYCLIC

PHOSPHODIESTERASE

STIMULATING

Received

AND

A.

ACTIVITY

Adamo

M.

FOLLICLE

AMP

Stefanini

Generale,

14, 00161

BY

CYCLIC

and

Embriologia

Scarpa

ADENOSINE

DIBUTYRYL

S.

1044-1050

Universita

Roma,

di

Roma

ltalia

1,198O SUMMARY

Total phosphodiesterase activity was measured in Sertoli cell culture after exposure to isobutyl-methyl-xanthine, dibutyryl cyclic AMP and FSH. After 24 hr of incubation both FSH and dibutyryl CAMP caused a significant increase in total phosphodiesterase activity of Sertoli cell homogenates (control: 66 2 16 pmoles/min/mg protein; FSH: 291 -+ 25 pmoles/ min/mg protein; dibutyryl CAMP: 630 + 70 pmoles/min/mg protein). FSH stimulation was potentiated by isobutyl-methyl-xanthine. Both in the presence and absence of xanthine, the indu ction of phosphodiesterase was dependent on the FSH concentration, with maximal stimulation achieved with 0.5-1.0 pg FSH/ml. The induction of phosphodiesterase activity by hormone was abolished by cycloheximide treatment. The data suggest that FSH regulates phosphodiesterasd activity via changes of CAMP levels in Sertoli cell in culture.

I NTRODUCT The

Sertoli

FSH

stimulates

and

consequently

specific to testosterone

ad

synthesis FSH

and

ccl

I

cyclic

dependent

dependent

this

factor

state

AMP

has

(8)

maturation,

the

is

by

reverted

been

proteins more Sertoli

hypophysectomy

recently, cell

such

(1).

ABP

synthesis

(10);

(6),

has

Sertoli

DNA

been

Copyright 0 198I by Academic Press, Inc. AN righfs of reproduction in an-v form reserved.

activation (2,5), activator inhibitor(g)

hormonal

suggested

ABBREVIATIONS : MIX, 3-isobutyl-1-methyl-xanthine; db-CAMP, 6-N,2-0-dibutyryl 3':5' monophosphate; CAMP, adenosine 3': 5' cyclic monophosphate; FSH. Follicle fluoride; BSA,Bovine Serum Albumin; ting Hormone; PMSF, phenyl methyl sulfonyl gen Binding Protein. 0006-291X/81/041044-07$01.00/0

no distal

kinase to

(2,

Although

synthesis

plasminogen

(1).

levels

cell,

include

protein

refractory it

the

and

of

becomes

(3).

These

protein as

AMP

activity in

total

agonists

cyclic

kinase

described ot

adrenergic

increases

demonstrated

been

stimulation

and,

and

protein

have

specific

testosterone activity,

dependent

cascade

of

FSH,

cyclase

(41,

secretion

inhibiting testis

for

phosphorylation

AMP

and

cell

adenylate

aromatization

During (1)

i

a target

enhances

cyclic

of

is

Sertol

FSH

events

(7),

cell

I ON

stimulatioti that

phospho-

adenosine StimulaABP Andro-

Vol.

98,

No.

diesterase

might

More

recently

becomes

it

play

an

has

been

insensitive

other

cell

cyclic

to

culture

AMP

also

have

that

in the

hormonal have

induces

the Sertoli

is

in

any

refractoriness

exposure On

the

agent

to other

that

phosphodiesterase turn

COMMUNICATIONS

this

after

(11). that

of

of

cell,

demonstrated synthesis

RESEARCH

maintenance

stimulation

activity

therefore

phosphodiesterase

Sertoli

further

role

BIOPHYSICAL

for

FSH

in

hand,

the

14)

vitro,

studies

increases

(12

responsible

(1).

in

intracellular and

that

the

refractory

state,of

(12).

We

dence

shown

phosphodiesterase

cell

AND

important

systems

levels

increased the

BIOCHEMICAL

4,198l

investigated activity

that

both cell

FSH in

and

operates dibutyryl

the

possibility

also

in

CAMP

that Sertoli

regulate

cells. phosphodiesterase

a

similar In

regulatory this

report activity

mechanism we

present in

the

of evi-

immature

culture.

MATERIALS

AND

METHODS

Sertoli cell enriched cultures were prepared from 15 day old immature Wistar rats following a procedure previously reported (2) with minor modifications(l5).The seminiferous tubule explants were maintained for 3 days in defined medium (Minimum Essential Medium, Eagle) at 32“C in an atmosphere of 95% air and 5% CO . On the third day of culture the exhausted medium was removed and the culture briefly ezposed to hypotonic buffer removes all the remaining germ cells adhering (Tris-HCl 20 mM) for 3 min. This treatment to the Sertoli cell monolayer (16,17). All treatments were started at least 24 hr after the exposure to hypotonic solution. FSH, dibutiryl-cyclic AMP,MIX were added to the culture by removing the old medium and adding fresh medium supplemented with the various compounds. In the experiments in which cycloheximide (10 pg/ml) was used, the inhibitor of protein synthesis was added 30 min before the addition of hormone. At the end of incubation the medium was removed by aspiration, cell monolayers rinsed 3 times with phosphate buffered saline (PBS) and the culture plates transferred to 4°C. Cells were scraped with a rubber policeman and transferred to an all glass homogenirer; the homogenization buffer was Tris-HCl 25 mM, pH 8.0, 10 mM /&ercaptoethanol and 2 mM PMSF. The cells were disrupted by 30 strokes of the pestle and the effectiveness of the homogenization was monitored by phase contrast microscopy. The cell homogenate was directly used in the phosphodiesterase assay. The phosphodiesterase activity was measured according to the method of Thompson and Appleman (18). The incubation mixture contained Tris-HCl buffer, 20 m3, pH 8.0, p-mercaptoethanol 10 mM, cyclic AMP (12.5 PM and approximately 100,000 cpm [ H]cAMP), CaC121mM, MgCl 5mM in a volume of 2OOp. The assay was performed by measuring the rate of CAMP hydrolcsis by sequential withdrawal of an aliquot of the incubation mixture from a master incubation at 7,14,21 and 28 min. The reaction was stopped by addition of 10 mM EDTA and the sample boiled for 1 min. The reaction products were separated as described elsewhere (18). Under these experimental conditions the rate of the reaction was linear for at least 30 min, and proportional to the amount of protein added. Protein was measured by a modificatiog of the standard Lowry procedure (19), using BSA as standard. [ HICAMP was from NEN; dibutiryl cyclic AMP, PMSF and adenine from Sigma; BSA was from Miles; MEM from Gibco; MIX from Aldrich. Rat FSH (NIAMDD-rat-FSH-Bl) was a generous gift of the NIAMDD rat pituitary hormone distribution program.

1045

Vol.

98,

No.

4,1981

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

FIGURE 1. Effect of different treatments on total phosphodiesterase activity in immature Sertoli cell in culture. Cell monolayers were exposed for 24 hr to fresh medium with the following additions: MIX (0.1 mM), db-CAMP (0.5 mM), rat FSH (10 pg/ml) MIX (0.1 mM) + db-CAMP (0.5 mM), FSH (10 pg/ml) + MIX (0.1 mM), cycloheximide (lOpg/ml). At the end of the incubation cell monolayers were processed and phosphodiesterase activity assayed as described in the Methods. Each bar represents the Mean + 1 SDM on three determinations on three different culture plates. Data are expressed as nanomoles of CAMP hydrolyzed/ min/mg protein of cell homogenate.

RESULTS The

effect

presented

in

ase

in

ved

when

tive

various

Fig.

1.

at

the

stimulated. enzyme

the

phosphodiesterase but

activity,

consistently

that

phosphodiesterase

effect

+

order

protein

was

used;

FSH

MIX

the

Sertoli

was

culture

the

of

10

was

effect

obtained

with culture

of

and

of

also the

combined

66

hr

larger

db-cAMP

did

Data

FSH -+

16

+

not

+

for

24

pmoles/min/mg

hr

obser: addimaxi*’

raising

was FSH

show

incre.

already

in

collected

MIX

was

being

of

an

effect

effective

effect

is

produced

gonadotropin

db-cAMF. with

value

24

A much MIX

activity

activity

pg/ml

The

basal

fold.

for

phosphodiesterase

the

the

phosphodiesterase medium

2

(F1g.1).

of

total

the

t&G

potentiated

byI

MIX

was

slight-

from

4

differen:!

enhanced

protein

the (Mean

-+

protein.

whether

with +

that

a

the

of

MIX

cell

combination

db-CAMP,

from

test

the

concentration

treatment

to

to

used,

than

pmoIes/min/mg

stimulation of

approximately

unlike

72

synthesis,

hormone

of

activity

597

In

activity

lower showed

to

MIX

a

but

Sertoli

of

inhibitor

experiments

on

concentration at

DISCUSSION

addition

mM)

FSH

tal

red

The

(0.5

&-cAMP

effects

SDM)

treatments

phosphodiesterase

mally

ly

of

AND

observed

cell

cycloheximide almost

stimulation

cultures

were (II)/.&mI).

completely

abolished

1046

of treated, Under (Fig.

phosphodiesterase 30 these 1).

min

activity before

experimental

and

requ-j

throughout condition

the

Vol.

98,

No.

4,198l

BIOCHEMICAL

0

AND

8

BIOPHYSICAL

16

24 Tome -

FIGURE ture : pg/ml) monolayers Methods. plates.

phosphodiesterase

a fast

increase

during

sively

slower

increase

up

The

stimulation

at

rase

inhibitor I more

se

of

MIX,

db-CAMP

sis

that

be and

the

therefore for

reported

can

by

Longer

lO&ml

FSH

with the

FSH

of

a

mentioned

was

of

0.2

the

in

half

were

dependent

&ml.

followed

not on

the

of

response,

response

FSH

+

MIX,

by

a

propres

concentration

enzyme

presence

maximal

FSH

studied.

the

The

maximal

with

21,

stimulated

increase

stimulation,

treatment

times

maximally

ED50

hr (Fig.

stimulation

alone

26

a

incubation

activity

3).

(Fig.

suggest

regulated the

FSH

phosphodiesterase and

synergistic

effect

of

phosphodiesterase

that

appropriate

that

by

induction

expected an

hr.

hr

phosphodiesterase

-

to

8

first

during

activity the

phosphodiest!

also being

(

rendered

produced

the at

a do-

pg/ml. data

cells

26

besides

sensitive

0.07 The

5

of

the to

medium

a dose

ccl

Ii

of

incubation

fold)

30

hr

studied

activity,

showed

the

COMMUNICATIONS

2. Time course of induction of phosphodiesterase activity in Sertoli cell in culeffect of FSH + MIX. Immature rat Sertoli cell cultures were exposed to FSH (10 and MIX (0.1 mM) for the indicated time intervals. At the end of the culture cell were processed and phosphodiesterase activity assayed as described in the Each point represents the Mean + 1 SDM of three determinations on three different Data were corrected for protein content.

Total

in

RESEARCH

any

period

other of

db-CAMP of

activity treatment.

MIX

and is

agent

time,

that

would

The

FSH

are

also

produce

that

exposure decrease

cultured

maximal

both

primarily would

of

stimulation

consistent

regulated raise

Sertoli an

immature obtained with

by cell

Serto-

the

hypothe-

CAMP

levels.

CAMP

concentration,

increase

of

phosphodiesterase

of

Sertoli

cell

enriched

in

phosphodiesterase

It

is

activity. Fakunding rous

tubules

et to

al. FSH

(20) for

have 20

min

reported produces

a

1047

5%

seminifeactivity.

This

4

Vol. 98, No. 4,198l

BIOCHEMICAL

0 ’

0

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

,!’ 0.01

0.1 FSH - pg/ml

1.0

10

FIGURE 3. Dose response study of the effect of FSH and FSH + MIX on phosphodiesterase Cell cultures were exposed for 24 hr to activity in immature Sertoli cell in culture. increasing FSH concentrations in absence and presence of 0.1 mM MIX. At the end of the incubation cells were processed and phosphodiesterase activity measured as described in the Methods. Each point represents the Mean of triplicate enzyme determinatiors on two different culture plates. The deviation from the mean was within 10% for all observations.

finding

is

events

could The

of

not

in

contrast

both

occur

relatively

slow

cycloheximide It is

rase

known

is to

unlike,

necessary

involved

or

more

worth

mentioning

the

same

cell

at

of the to

complex

describe

a

different

enhancement

of

determine

whether

events

are

delayed

effect

of

FSH:

the

two

times.

phosphodiesterase

in of

that

in

the

calmodulin

a protein a cell

sphodiesterase Stimulation fibroblasts

enzyme

at

this

regard,

kinase free

observed

activity

and

activity

requires

actual the

base

the

synthesis

activity

remains

of

phosphodiesterase

in

(21)

increase

On

inhibitor system

that

activity

levels.

(12-13) experienced

in

phosphodiesterase

therefore,

least

mulation

that

that

be

stimulate

increase

for

data

the

inhibitory de

novo

phosphodiesterase of

the

effect protein-

de

increased

novo

sync

phosphodieste-

activity. It

sis

will

our

induction

suggest

synthesis. thesis

in

with

(23). to

in

the

contrary

(9)

which

also

Th e

effect

of

be

defined.

myoblasts

our

study

(up

to

in

12

1048

fold)

has

under

FSH

this

shown

&-cAMP

to

on

and

higher

the than

it

is

depends

on

the

synthe-

activity the

at

induction

has ampl

that

factor

(22):

increase

xanthine

however is

control

phosphodiesterase regulation

culture,

a

activity

been

inhibits

by

(14)

not

calmodulin,

phosphodiesterase FSH

activity

and

is

of

itude observed

of

been

pho-

reported of

the in

st ithose

Vol.

98,

No.

systems.

It

cultured

for

or

is

worth

4

&-cAMP

described

in

dependent

Sertoli

forms se

view (1)

is

days

in

the might

In

noticing a

AND

that,

BIOPHYSICAL

unlike

chemically

RESEARCH

fibroblasts

defined

and

medium

COMMUNICATIONS

myoblasts,

lacking

serum

Sertoli or

FSH,

cells prior

were

to

hormone

stimulation.

Finally, activity

BIOCHEMICAL

4,198l

limited

be

a

Sertoli

to

catabolism

consequent

phenomenon

(11).

cells

the

will

CAMP

relevant

celt of

it

increased

to

Whether

unresponsiveness finding

also a particular

that be

the

the

to

induction

this

mechanism

remains

to

Sertoli

interesting

to

cell verify

increased of is

be

a short also

term

refractory

involved

in

the

state age

determined.

contains whether

phosphodiesterase

multiple the

induction

phosphodiesterase of

iso

phosphodiestera-

isoenzyme.

ACKNOWLEDGMENTS This work was supported by C.N.R. project "Biology of Reproduction", grants No 79.01166. 85 and No 79.01176.85 and by Ford Foundation grant No 790-0659. The authors are indebted to Mr. L. Petrelli for his skillful1 technical assistance and to Rat-Pituitary Hormone Distribution Program (NIAMDD) and to Dr. A.F. Parlow for the generous gift of rat FSH.

REFERENCES 1. Means, A.R., Dedman, J.R., Tash, J.S., Tindall, D.J., van Sickle, M , and Welsh, M. J. (1980) Ann. Rev. Physiol. 42, 59-70. 2. Dorrington, J.H., Roller, N.F.,and Fritz, I.B. (1979) Mol. Cell. Endocrinol. 3, 57-70 3. Fakunding, J.L., and Means, A.R. (1977) Endocrinology 101, 1358-1368. 4. Dorrington, J.H., Fritz, I.B., and Armstrong, D.T. (1976) Mol. Cell. Endocrinol. 6, 117-122. 5. Griswold, M.D., Mably, E.R., and Fritz, I.B. (1976) Mol. Cell. Endocrinol. 4, 139-149 6. Fritz, I.B., Rommerts, F.G., Louis, B.C., and Dorrington, J.H. (1976) J. Reprod. Fert 46, 17-24. Lacroix, M., Smith, F.E., and Fritz, I.B. (1977) Mol. Cell. Endocrinol. 9, 227-236. 8. Steinberger, A., and Steinberger, E. (1976) Endocrinology 99, 918-921. 9. Tash, J-S., Dedman, J.R., and Means, A.R. (1979) J. Biol. Chem. 254, 1241-1247. 10. Fritz, I.B., (1979) Biochemical Action of Hormones, vol. V, 249-281, Academic Press, New York. 11. Verhoeven, G., (1980) J. Steroid Biochem. 12, 315-322. 12. Manganiello, V., and Vaughan, M. (1972) Proc. Natl. Acad. Sci. USA, 69, 459-462. 13. Russel, T.R., and Pastan, I.H. (1974) J. Biol. Chem. 249, 7764-7769. 14. Ball, E-H., Seth, P.K., and Sanwal, B.D. (1980) J. Biol. Chem. 255, 2962-2968. 15. Palombi, F., Ziparo, E., Rommerts, F.G., Grootegoed, J.A., Antonini, M., and Stefanini, M. (1979) J. Reprod. Fert. 57, 325-330. 16. Ziparo, E., Geremia, R., Russo, M.A., and Stefanini, M. (1980) Am. J. Anat.(in press) 17. Stefanini, M., Ziparo, E., Palombi, F., Galdieri, M., Geremia, R., and Russo, M.A. (1980) Animal Models in Human Reproduction, pp. 149-157, Raven Press, New York.

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Thompson,

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A., J.L.,

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M.M.

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J.R.

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COMMUNICATIONS

311-316.

Biochem. 70, 24l.-250. Mena, C-R., and Means,

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