Vol.

63,

No.

4, 1975

BIOCHEMICAL

AND

BIOPHYSICAL

MESSENGER RNA SYNTHESIS IN RATS: EFFECT OF GONADOTROPINS

P.R.K. Laboratory Biology, Medical

Received

February

THE

RESEARCH

TESTIS AND

COMMUNICATIONS

OF IMMATURE CYCLIC AMP

Reddy and C.A. Villee of Human Reproduction and Reproductive Department of Biological Chemistry, Harvard School, Boston, Massachusetts 02115.

25,1975

SUMMARY Two hours after the introtesticular injection of FSH, hCG or cyclic AMP, the incorporation of labeled uridine into poly(A)-rich RNA was increased. Pretreatment with actinomycin D inhibited the incorporation of uridine into mRNA. After the seminiferous tubules and interstitial cells were separated by treatment with collagenase, FSH treatment increased mRNA synthesis only in the tubules whereas hCG stimulated mRNA synthesis only in the interstitial cells. Cyclic AMP increased the synthesis of mRNA in both interstitial c-lls ai-d seminiferous tubules. These results suggest a differential action of the two gonadotropic hormones in the c-lls of the testis; both effects appear to be mediated by cyclic AMP.

-INTRODUCTION Gonadotropic rat

(l-5).

An

treatment

increased

with

to the

hormones

shown

that

the

hormones

was

the

end

nism

studied. of action

Abbreviations Gonadotropin;

Copyright All rights

in the

from The

oligo

testis

of immature

experiments

of gonadotropic

used: cyclic

AMP

(dT)

animal

columns

(15).

rats

in the

was

observed

testis

contains

RNA

can

In the

following

designed

hormones

o I9 75 by Academic Press, Inc. of reproduction in any form reserved.

RNA

be

on the

Stimulating Adenosine

1063

and

D (3,4).

our

RNA

isolated

FSH,

understanding

after in response it was

sequence

reported

with

of the

Recently

conveniently

experiments

testis

shortly

a polyadenylate

treatment to increase

in the

of proteins

octinomycin

origins rich

FSH - Follicle AMP - Dibutyryl

and

synthesis

with

poly(A)-

were

of proteins

Tf le increased

by pretreatment

using

The

synthesis

of cyclic

R“JA

(11-14).

the

(6-10).

inhibited

messenger

chromatography

was

production

gonadotropins

3’ terminal

of mRNA

stimulate

hCG,

here

at

by affinity the

synthesis

or cyclic of the

mecha-

testis.

Hormone; 3’:5”

hCG - Cyclic

- Human Chorionic Monophosphoric

Acid.

AMP

Vol.

63,

No.

4,1975

MATERIALS

BIOCHEMICAL

AND

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

METHODS

Immature 2l-doy-old rats (Sprague-Dawley strain) were obtained from Chorles River Breeding Laboratories. Deoxyribonuclease (RN&e free) and collagenase I were purchased from Worthington Biochemicals. Dibutyryl cyclic AMP and actinomycin D S-IO) was generously provided by the NIH and were obtained from Sigma. FSH ( ovine hCG was obtained from Ay rst Laboratories. Oligo (dT) cellulose was obtained from Collaborative Research. -uridine (46 ci/mmole) and Aquasol were purchased I33H from New England Nuclear. All other chemicals were of analytical grade. Rats were injected with 100 pg of FSH or cyclic AMP or with 100 JU of hCG in 50 pl of 0.15 M soline per testis using a Unimatrix syringe with a 27 gauge eedle. Control animals were injected with 50 pl of 0.15 M saline. Simultaneously, -uridine rf33H (2 pc) was injected into each testis. In some animols actinomycin D (100 ug) was injected intraperitoneally two ho&s before the introtesticular injection of hormones or cyclic AMP. Animals were killed by decapitation two hours after the treatment. Decapsulated testes from six to eight animals given each treatment were pooled ond rapidly washed in cold saline. RNA was extracted using 9 ml of 20 mM sodium acetate buffer and I ml of loo/c sodium dodecylsulfate in cold phenol (16). Th e f inal RNA precipitate was dissolved in 0.3 ml of distilled water and the optical density was measured at 280, 260 and 230 nm in a Zeiss PMQ I I spectrophotometer. Groups of decopsulated interstitial cells and seminiferous two types of cells as described

testes were tubules. above.

incubated in collagenase (17) RNA was extracted separately

to separate from the

Poly (A) RNA was separated from the total RNA extract using an oligo (dT) column (15). A known amount of RNA was applied on the column. High salt buffer, 0.5 M KCI - IO mM tris-HCI, pH 7.4 was used to elute all of the RNA which was not bound to the column (subsequently termed “unbound RNA”). The bound poly (A) RNA was eluted in 6 ml of IO mM tris-HCI, pH 7.7. After recording the absorbancy of unbound and poly (A) RNA at 260 nm the radioactivity was measured in an aliquot using a Packard scintillation spectrometer and a mixture of Aquasol-water-acetic acid (870:80:50) as scintil lotion fluid. Lineor 5 -200/c sucrose gradients were prepared in IO mM tris-HCI buffer (pH 7.4) with the aid of a Buchler gradient former. Unbound RNA, 200 pg, or poly (A) RNA, 50 pg, was concentrated to 0.1 ml in an Amicon Concentrator and layered over the gradients. The gradients were centrifuged for 210 min at 45,000 r.p.m. in a Spinco SW 50 rotor at 4OC. Following centrifugation the bottom of the tube was punctured, IO drop fractions were collected in scintillation viols, and the mdioactivity of each fraction was measured. RESULTS

AND The

fractions cyclic than

incorporation in the

AMP. inthe

DISCUSSION of radioactive

testis

increased

The

increased

unbound

RNA,

uridine

significantly incorporation indicating

into following

was a preferential

1064

greater

both

unbound

and

treatment in the stimulation

with poly

(A) of

the

poly

(A)

RNA

FSH,

hCG

RNA

fractions

synthesis

or

of

Vol. 63, No. 4,1975

TABLE

1.

BIOCHEMICAL

E

OF FSH, URIDINE

hCG INTO

AND BIOPHYSICAL

AND CYCLIC UNBOUND

RESEARCH COMMUNICATIONS

AMP ON AND POLY

Unbound

THE INCORPORATION (A) RNA OF THE

RNA

Poly

OF TESTIS. (A)

RNA

Saline

1140

3370

FSH

1415

8615

hCG

I.520

7880

I360

5450

Cyclic

AMP

Saline

+Actinomycin

FSH

+Actinomycin

hCG

t Actinomycin

Cylic

AMP

D D D

+Actinomycin

D

Testes from six to eight cpm/lOO pg RNA, are

TABLE

2.

animals were used in each the means of three experiments

EFFECT OF FSH, hCG AND OF c3H] URIDINE INTO INTERSTITIAL CELLS.

Seminiferous unbound 760

FSH

II40

hCG

synthesis whereas

hCG

468

experiment. for each

The data, treatment.

AMP ON THE SEMINIFE!?OUS

poly

expressed

INCORPORATION TUBULES AND

Interstitial

(A)

RNA

as

unbound

Cells

RNA

poly

(A)

550

790

1520

840

4500

870

2100

640

1540

were used in each of three experiments of the

and

poly

(A)

(A)

RNA

in the

stimulated

390

460

Pretreatment

of poly

434

5400

from six to eight animals pg RNA are the means

unbound

580

590

Testes cpm/lOO

of both

396

480

AMP

molecules.

460

1410

Cyclic

these

418

Tubules

RNA

Saline

CYCLIC RNA IN

514

the

RNA

rat

in all

with

synthesis

actinomycin

animals

seminiferous

experiment. for each

of poly

D inhibited

(Table

tubules (A)

1065

RNA

The data, treatment.

I). but

not

in the

FSH

expressed

the

synthesis

stimulated

in the interstitial

the

interstitial cells

cells but

RNA

as

Vol.

63,

not

No.

in the

both

BIOCHEMICAL

4,1975

seminiferous

seminiferous

employed FSH

tubules. tubules

produced

Cyclic

and

AND

BIOPHYSICAL

AMP

stimulated

interstitial

a smaller

cells

increase

3H I3

(Table

in RNA

RESEARCH

2).

uridine

The

synthesis

than

density

gradient

COMMUNICATIONS

dose those

of

uptake cyclic

in AMP

observed

with

or hCG. Analysis

three

of the

distinct

peaks

unbound (Figure

RNA

by sucrose

I) whereas

poly

(A)

RNA

from

centrifugation

the

testes

revealed

of both

control

600

A.

UNBOUND

4

IBS

500

---

1

,-. ,’

‘\

(I’

)I--:

I

\

,’

‘\

\.-*’

:

Iv

bOortom

and

FSH

Sucrose

density-gradient

treated

with

treated

rats

working

hypothesis

to their

receptors

as to increase

was

and RNA

saline

there

synthesis,

,’

: :

L’ .

'\ '\

5

IO

that the

2b

IS

2’5

3b top

FRACTION NUMBER

pattern

of

unbound

and

poly

(A)

RNA

from

The

current

animals

or FSH.

distributed

suggests

‘, !

I

O-!

I.

:

i

,r\

20

FIGURE

I

RNA

‘\\ :30 ti

RNA

SALINE FSH

in a broad steroid

region

hormones

steroid-receptor especially

are complex

the

synthesis

1066

around taken

18s. into

the

regulates of poly

(A)

nucleus nuclear

RNA

bound events

(16,18).

so The

Vol.

63,

No.

present

results

poly The

(A)

RNA

synthesis

Recently

BIOCHEMICAL

4, 1975

demonstrate

that

in the

and

of

testis

nuclear

Abney

et al

testes

of hypophysectomized

In our

studies

FSH

tubules

while

hCG

hCG

(22)

thesis

of poly

(20)

was

been

(A)

Cyclic cells

(24)

in both

of nuclear

RNA

polymerases.

protein

kinase

from

calf

evidence may

AMP

which role

that the

cells The two

appear

gonadotropin

the

cyclic of

our

of hCG

AMP

to be

in this

are

A cyclic

AMP

of RNA

AMP

stimulation

RNA

polymerases. FSH

This work was supported by a grant HD 1232 from the National Institute

yet

LH.

seminiferous

binding

of

seminiferous

experiments

of the

evidence

(A)

uterus

syn-

of

the

testis.

presence

the

(A)

RNA

the

in messenger

RNA

in the

of many

synthesis

and

World Health Health and

1067

Organization and Human Development.

testis

workers

in response of events the

precise

clear.

from the of Child

This

of cyclic

testis

sequence

isolated

II (26).

synthesis

of

dependent

kinase

in the

precise

RNA syn-

AMP

accumulation

synthesis

(A)

stimulation

lb and

observations

concomitant

RNA

to the

protein la,

in bone

increased

of a cyclic

dependent

and

of poly The

to be due

The

(23)

synthesis

polymerases

However,

results

the

of poly

and

of poly

interrelated.

not

and

in the

present

in the

appears the

and

in the

FSH

The

is direct

of

d emonstrated

observation

administration AMP

cells

to cyclic

rat.

(19).

in the

to receptors

increased

interstitial

(25)

of

cells.

synthesis

AMP

phosphorylation

activation

and

and

earlier

localized

interstitial

in the

RNA

cyclic

response.

testis.

stimulate

of the

was

compartments

in the to

synthesis

FSH

this

of

of polyribosomes

a combination

of

synthesis

observed

with

demonstration different

was

stimulation

and

(21)

the

D inhibits

treatment

in the

COMMUNICATIONS

stimulate

actinomycin

RNA

effective

et al

binding

testis

agents

of cyclic

(A)

studies

testis

stimulated

differential

to these

poly

in response

in the

from

in the

of

tubules

Reddi

suggests

on the

treatment

shown

In our

RNA

ovary

result

after

RESEARCH

AMP

increased

rats

in these

seminiferous

thesis

reported

interstitial

was

--in vitro.

the

to FSH

transcription AMP

cyclic

in response

observed.

gonadotropin-induced

and

hCG

with

similarly

RNA

BIOPHYSICAL

pretreatment

have

in the

has

FSH,

stimulation

to receptors

tubules

RNA

AND

by grant

by

BIOCHEMICAL

Vol. 63, No. 4,1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES I.

Means,

A.R.,

and

Hall,

P.F.

(1967)

Endocrinol.

81,

2.

Means,

A .R.,

and

Hall,

P. F.

(1968)

Endocrinol

. 82,

3.

Means,

A.R.,

and

Hall,

P.F.

{1969)

Biochemistry

4.

Goswami,

5.

Villee, D. and ed. Niu, M.C.

6.

Murad,

7.

Hollinger,

8.

Kuehl, F.A., 2, 154-163.

9.

Dorrington, Commun.

A.,

F.,

Skipper,

Strauch,

S.,

T.,

and

(1970)

IO.

Braun,

and

II.

Lim,

12.

Dronell, J.E., 68, 1321-1325.

13.

Lee, 68,

14.

Edmonds, Sci. USA

15.

Aviv,

16.

Villee,

C.A.

(1975)

17.

Moyle,

W.R.,

and

18.

Villee, C.A., (in press).

19.

Means,

A.

20.

Abney, 3956-3962.

T.O.,

21.

Catt, (1974) M.L.,

M., 68,

4293-4298.

W.L.

(1968)

S.

Canellakis, Wall,

R.,

Vaughan, 1336-1340.

and

and

Tushinski,

and

P. J.

Steroid

Skipper,

J.

89, J.K.,

and

94,

108,

147-152.

and Development, York. Acta

177,

591-598.

Biol.

Reprod.

Biophys.

Res.

(1973) Reddy,

Proc.

Nat.

Proc.

Nakazato,

(in

Biochem.

710-712.

(1971)

H.

Acad.

(1970)

1028-1033.

(1971)

G.

J.L.

(1972)

227,

Nat.

Biochem.

and

Biophys.

Humes,

I .B.

R.J.

and

Proc.

A.,

Endocrinol.

Nature

Jr.,

Ramachandran,

and

Fritz,

Brawerman,

(1972)

J.

in Reproduction Elsevier, New

Biochim.

Endocrinol.

(1970)

Grigorescu,

(1971)

(1974)

M.H.,

Leder,

J.,

and

E.S.

J.,

(1969)

Biochem.

533-540.

Tarnoff,

R.G.,

Sepsenwol,

M.

9,

D. J.,

S.Y., Mendecki, 1331-1335.

H.,

Sci.

J.H., Vernon, 1523-1528.

and

8,

Williams,

Vaughan,

Life

Panatelli,

46,

and

597-602.

Goswami, A. (1973) The Role of RNA and Segol, S.J., pp. 73-85, American

M.A.

L.,

J.K.,

1151-1160.

Sci.

Acad.

Nat.

(1971)

USA

Sci.

Acad.

Proc.

69,

USA

Sci.

Nat.

USA

Acad.

1408-1412.

press). J.

Endocrinol.

93,

127-133.

P.R.K.

(1975)

J.

Steroid

W.L.

(1974)

Biochemistry

Biochem.

981-989. Williams,

K., Tsuruhara, T., Mendelson, C., Ketelslegers, J.M., Hormone Binding and Target Cell Activation in the Testis, and Means, A.R,, pp. l-30, Plenum Press, New York.

1068

and ed.

13,

Dufau, Dufau,

M.L.

Vol.

63,

No.

22.

Bhalla,

23.

Sharma,

24.

Peck,

4, 1975

BIOCHEMICAL

V.K., S.K., W.A.,

and

Reichert,

and

Talwar,

Messinger,

AND

L.E., G.P. K.,

BIOPHYSICAL

Jr.

(1973)

(1970)

J.

Kimmich,

G.,

J. Biol. and

RESEARCH

Biol.

COMMUNICATtONS

Chem.

Chem.

249,

245,

Carpenter,

43-51.

1513-1519. J.

(1974)

Endocrinol.

95, 289-298. 25.

Reddi,

A.H.,

Ewing,

L.L.,

and

Williams-Ashman,

H.G.

(1971)

Biochem.

J.

333-345. 26.

Jungmann,

R.A.,

Hiestand,

P.C.,

and

Schweppe,

249, 5444-5451.

1069

J. S. (1974)

J.

Biol.

Chem.

122,

Messenger RNA synthesis in the testis of immature rats: effect of gonadotropins and cyclic AMP.

Vol. 63, No. 4, 1975 BIOCHEMICAL AND BIOPHYSICAL MESSENGER RNA SYNTHESIS IN RATS: EFFECT OF GONADOTROPINS P.R.K. Laboratory Biology, Medical...
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