Early effects of retinol and retinoic acid on protein synthesis in retinol deficient rat testes

Vol. 151, No. 1, 1988 February 29, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 53-60

EARLY EFFECTS OF RETINOL AND RETINOIC RETINOL Riaz

DEFICIENT

University

January

and Medicine

School

Nashville,

IN

Chytil

of Biochemistry

Vanderbilt

PROTEIN SYNTHESIS

ON

RAT TESTES

ul Haq and Frank

Departments

Received

ACID

of Medicine

TN 37232

7, 1988

SUHHARY: When dn ["S] labeled mixture of methionine and cysteine was injected intratesricularly into retinol-deficient rats, two hours later more than 980 cytosolic proteins were detected by computer aided two dimensional two hours after oral refeeding retinyl electrophoresis. Furthermore, gel acetate as the source of retinol to retinol deficient rats, synthesis of 286 proteins was inhibited and that of 101 proteins was activated. Refeeding with retinoic acid leads in two hours to even higher inhibition of protein synthesis and the labeling patterns of proteins are not identical when compared to retinol refed rats. The results indicate that retinol or retinoic acid quickly influence expression of many proteins and suggest that retinol action in the testes is not identical to that of retinoic acid. 0 1988 Academic Press, Inc.

Retinol Animals

plays

fed

whereas

an

a diet

lacking

Sertoli

cells

little

affected

(1).

cells

eventually

Experiments (5)

genomic

expression.

events

of

answer

acid

on of

or specific

retinoic

influenced

deficient

rat

proteins?

with

retinol

Thirdly, or

the

(for

action Firstly, synthesis?

by two hour does retinoic

acid

reported

protein

what

are

such

short

acid

influence

term

influence

In

the

some of

of

retinol

refeeding synthesis

early

We used cytosols

effects how

work

the

synthesis. the

of

to

retinol

extensively

is

or retinoic of a retinolof the

same or

0006-291X/88 53

in

to be activated

testicular

Secondly, refeeding

(~$3). cells

see 6).

of

to be germinal

functionally

we describe on

appear

restored,

retinoic

review

(1).

epithelium,

teratocarcinoma

were

electrophoresis

protein

is

metabolite

rat,

acid

questions.

rat?

different

acid

testes

germinal

and with

the

cells

retinol

genes

retinol-deficient

dimensional

proteins

its

the

interstitial of

or

forty

testicular

to deficient

(4)

retinol

or

following

deficiency

animal

of

of

morphologically

or retinoic

two

and

supply

that

the

retinol

the

synthesis

using

atrophy

present

About

by retinol

aided

computer

still

whole

differentiation

show

When dietary

indicate

here

in

retinol

are

the

culture

reported

role

regenerate

with

or repressed

essential

$1.50

Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 151, No. 1, 1988

NATENIALS

AND BIOPHYSICAL RESEARCH COMMUNICATIONS AND mTNoDs

a mixture of [""S]-labeled methionine and Materials - Trans ['5S]-label, cysteine 70:20 (specific activity 1100 Ci/nnnol), was purchased from ICN, Irvine, CA. Sprague-Dawley rats were obtained from Sasco Company, St. Louis, MO. Intragastric Feeding and Labeling of Testicular Proteins - Animals - Rats (21 days old) were made retinol deficient as described previously (7). Serum retinol level of deficient rats was determined by fluorometric method (8) and ranged between 2-4 ng/lOO ml, indicating that the rats were retinol deficient indeed. Retinol-deficient animals were fed 0.2 ml of cottonseed oil alone or with 100 ng of retinyl acetate as the source of retinol or retinoic acid by intubation into the stomach (4). Chow fed rats were used also as retinol Ten sufficient again after gastric intubation of 0.2 ml of cottonseed oil. minutes later, the rats were lightly anesthetized with ether. Next, the right or left testicle was exposed by a small incision and injected with 0.9 mCi of Trans ["?S] label using a Hamilton syringe. The incision was stitched immediately after the injection and the rats were allowed to recover. After two hours, the animals were killed by decapitation, blood was collected for retinol determination, and the injected testicles were excised. Preparation of Cytosols - Testes were homogenized in 1OmM Tris HCl, 0.25 M sucrose, pH 8.5 buffer (1:6 w/v) with a Potter-Elvejhem homogenizer and the homogenate centrifuged at 105,000 x g for one hour to prepare cytosols. Two Dimensional Gel Electrophoresis - Cytosols were treated according to the method of Garrels (9). Briefly, 400-500 ng of cytosolic proteins were diluted with SDS sample buffer (9.95 M urea, 0.3% SDS, 100 mM dithiothreitol and 4% NP-40) to give a protein concentration of 2 ng/nl, heated in boiling water for two min, then cooled on ice. 10 ~1 of DNA-RNase solution (1 mg DNase, and 0.5 mg RNase/ml per 200 ug of protein) were added next. Samples were then chilled on ice for two min with occasional vortexing and snap frozen in liquid nitrogen. Two dimensional gel electrophoresis has been standardized, and up to 3000 labeled proteins can be separated and detected on a single autoradiograph (10, The computer scanning 11). system developed by Protein Data Base Inc. (Huntington Station, NY) can detect l/10 of dpm. The samples were analyzed using gels containing pH 3-10 ampholines in first and a 15% acrylamide in second dimension. All samples were loaded at 100,000 dpm/gel. Dried, "Enhance" (New England Nuclear) treated gels were exposed for 10 days for autoradiography. Films were then scanned using a computer program which automatically detects and integrates the densities of spots. The molecular mass of proteins analyzed ranged between 10 kDa to 120 kDa. The computer analysis provided quantitative measurement of incorporation of radioactivity into proteins. The results of gels of cytosolic proteins from testes of retinol deficient rats were used for initial coordinates to match protein spots from testes of rats fed chow, refed retinyl acetate or retinoic acid. RESULTS AND DISCDSSION Protein

Synthesis

experiments mCi

of

in

have

shown

[Ts]

trichloroacetic

label, acid

electrophoresis. were

injected

shows

labeling

Using

computer

the

Testes

that

two

hours

sufficient

this

patterns analysis

dose of

Retinol-Deficient after

proteins

to

experiments, and

testicular

983 protein

proteins spots 54

perform

proteins were

in

Preliminary

incorporated two

dimensional

retinol-deficient were

detected

injection

was

therefore,

cytosolic

Rats

intratesticular

radioactivity

precipitated

In subsequent with

of

analyzed.

retinol (Table

deficient 1).

of

0.9 into gel

rats Fig. 1 rats.

Vol. 151, No. 1, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

150. 90. 70, 50. 40, 30,

r6bX

20.

2 5

18-m

4,95

4to

5.90

6,85

7.8

PI Figure 1. Fluorography proteins from retinol-deficient for relative quantitation identification numbers. were electrophoresed as

Retinol Proteins proteins, labeled

TABLE

or

Retinoic

Acid

- To study

of

dimensional gels of ["'S] labeled cytosolic testes. Protein spot #1 was used as standard of proteins. Some protein spots were assigned About 20 ug of cytosolic proteins (100,000 dpm/gel) described in MATERIALS AND METHODS.

Repress

effects

retinol-deficient amino

acids

were

Synthesis 1. Effect Rats.

of of

two

and

of

retinol

rats

were

injected

Activate on synthesis refed

intratesticularly

Cytosolic Proteins Short Term Retinol

Synthesis

of

retinol

for

Number Synthesis

of of

of

cytosolic

two

and

hours

Retinol-deficient Refeeding

Proteins Which

Inhibited

Was Activated

983

Retinal-deficient retinol refed'

772

286

101

698

364

111

' Retinol-deficient intragastrically

refed'

and

rats were refed 100 pg of killed two hours later.

5.5

retinyl

acetate

or

["'S]

in Materials

Retinol-deficient

Retinol-deficient retinoic acid

Many

testicular

as described

in the Testes of and Retinoic Acid

Numberof Proteins Detected

AllimalS

Quickly

retinoic

acid

BIOCHEMICAL

Vol. 151, No. 1, 1988

Methods.

and

followed

As can

by extensive

instance,

772

represents

a 22 per

to

the

testes

of

retinol

of

1).

The testes

refeeding

(Protein

synthesis

of

fed,

individual

protein

a drop is

to

synthesis

in

(# the

retinol-deficient eventually

is

supported

by our

in

of

chow

rats.

testes

agreement

with

the

was evaluated

labeling that

these

see

Fig.

14,15,16)

fed

earlier

could

may

occurring

be

rapid

observation

that of

590

2.

retinol

It

restore

only

decline

after

decrease

protein

testes

is

retinol

of

therefore on protein

the

synthesis

of

animals.

678 proteins in

enough, in

in retinol-sufficient

observation

retinol-

be detected of

(see

in

Interestingly

shown).

were synthesis

in

testicular

6,85

7,8

This labeled is poly

Fluorography of two dimensional gels of testicular cytosolic after two hours of refeeding retinol-deficient rats with retinyl acetate. The horizontal arrows show spots, the intensity of which did not change relative to those detected in retinal-deficient testes. The upward and downward arrows point to the spots that have decreased or increased Conditions respectively relative to proteins from retinal-deficient testes. of electrophoresis as in Fig. 1.

proteins

56

of of

was observed

2).

to

change

consequence

detected

effects

This compared

synthesized

not

not not

inhibitory

4,o Figure

1 and

(data

early

The

were

scan.

as the

are

is

In this

When the

286 proteins

which

proteins. proteins,

proteins).

of

refeeding

computer

labeled

spots

testes

contention

the of

(983

animals that

by

rats the

16,

1, retinol

of these

number

some proteins

some proteins

to a degree

the

in

# 15 and

Table

synthesis

of

so pronounced

assume

2 and

detected in

retinol-deficient

retinol-sufficient

reasonable proteins

were

decrease

inhibition

deficient

Fig.

in the

spots cent

refeeding,

Table

from

alterations

protein

radioactivity

chow

be seen

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in A-

BIOCHEMICAL

Vol. 151, No. 1, 1988 containing

RNA content

retinol

to deficient the

lesser For

instance,

Fig.

1 and

selected the

of

activation

this

effect

2.

For

synthesis

synthesis

be seen

from

Table

others

1,

(101

can be seen

oral

retinol

administration

spot

# 17, was not

also

activated

No.

of

of

Fig.

286

of protein

Using

# 17 in 12 dpm was

this

On the

by retinol

not to a

inhibited).

1 showing

1.0.

2.6-fold.

affected

causes

concurrently

versus

inspection 1 in

a value

increased

refeeding

but

by visual

and assigned

of some proteins

after

of many proteins, of

protein

hours

calculation,

other

refeeding

hand,

(Fig.

the

1 and 2

# 1 to 10). Retinoic

acid

affect

Fig.

3,

that

of

the

refeeding data is

few

also appears

synthesis

of

activated

synthesis

presented affected

of

acid

and

quickly to

retinol

(6).

and has been

We have, found,

more

therefore,

as shown

affects

be

364 proteins. here

very

products

retinoic

acid acid

metabolite

gene

with

retinoic Retinoic

inhibiting

proteins

an obligatory

rats

proteins.

The

is

synthesis

retinol-deficient

acid

two

(4).

quantitation,

standard of

or

synthesis

the

as the

proteins to

as can

inhibition extent

hour

animals

Furthermore, only

one

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

than

to retinol

refed

Table

synthesis

effective

Similar

in

1 and

of

many

retinol

by

action,

retinoic

many

cytosolic

of 111 proteins. indicate

quickly

4195

that

by retinol

synthesis or

5190

retinoic

of

acid.

The

labeled rats with AND METHODS.

cytosolic retinoic

6185

PI Figure proteins acid.

shown

Fluorography of two dimensional gels of 3. after two hours of refeeding retinal-deficient For details see legend to Fig. 2 and MATERIALS

["5S]

total

Vol. 151, No. 1, 1988

number even

of

BIOCHEMICAL

cellular

higher

proteins

as these proteins

contrast

to

the

proteins

with

exception

several

hours

(13-15,16).

may be due in

to

influenced

compounds

and membrane

(6).

with of

use

of

among

various

cell

in

contrast

to

retinol Refeeding the

acid

retinoic

generated of

an identical

manner.

Table

retinol

labeling

of

cytosolic

already

shown

that

different shown

in here

retinoic

acid

2.

Are of

from

Not

place

in

culture.

It

Identical

retinol, of

retinoic

Spot"

20 21 22 23 24 25 26

Fig.

l234-

Relative quantity 12 dpm (see Fig. Protein numbering Retinol deficient acid intragastrically N-D. non detected.

most

occur

in

response

the

proteins

also

due to

testes

quite

of

the

possible tissue

that

to tissue

to

address

studies

They on the

from

animals also

PI

Retinol Deficient

5.93 6.96 7.57 7.65 7.68 7.55 4.00 7.55

28.5 10.3 7.8 6.8 20.6

1.9 10.2 6.5

due

in

the

summation

in

different

effects

on

laboratory

A-containing

have RNA were

Thus

the

indicate

that

retinol

of

Retinol Refed3

15.8 4.7 4.0

1.8 N.D.4 2.3 5.4 5.7

results and

some proteins

on the

Quantity of Radioactivity1

Synthesis

which

of

Incorporated

Retinoic

Acid3 Fed

30.8 11.4

15.1 12.4 19.8 5.8 16.0 9.8

of radioactivity is based on protein spot # 1 showing 1) which was given an arbitrary value of one. refers to their position in Fig. 1. 2 and 3. rats were refed 100 pg of retinyl acetate or retinoic and killed two hours later.

58

to

question,

(4).

synthesis

- As

compounds

this

of poly

be

the

by these have

Acid

could

3 allowed

refeeding

observations.

of Retinoic

acetate

2 and

Previous

mass

21.3 25.0 51.9 50-S 41.0 66.4 17.0 12.0

of to

the

is

Effect of Retinol and Retinoic Acid SOme Cytosolic Testicular Proteins

Molecular (b)

19

in

influenced

Relative

Protein

apparent

in temporal

Those

retinyl is

acid

a common effect

Differential

in

described

to cell,

we attempted

proteins

proteins.

exert

cell to

refeeding

Comparison

these

are

to label

takes from

cytoskeletal

and probably

which

be

communications.

testicular and

were

reports,

may vary

also

must

expression

radioactivity

cells

acid

animal

where

difference

translation patterns --in vitro retinol and retinoic acid refed

confirm

TABLE

effects

effects

synthesis 2 that

the

Retinol

described acid

whether

of

this

previous

types

on intercellular

Effects

above

to

whole

culture (12)

for

retinoic

to alter

using in

RESEARCH COMMUNICATIONS

or

described

specific

as compared

and retinoic

retinol

results

cells

higher

interplay

and may depend

been

The reason

experiments animal

by

transglutaminase

our

whole

have These

studies

the

AND BIOPHYSICAL

Vol.

151,

No.

may be mediated Figs. of

2 and

of

is

three

acid

synthesis

proteins

activated

by

proteins

refed

unaffected

19, retinoic

of proteins

retinol

refed

effects

are

retinol

(see 20,

and

Table

in II

and 23 also acid

refed

These

COMMUNICATIONS

set

acid.

retinol

refed

proteins

rats

in

(Table

22,

and

retinol

2). that

refed

in

25).

refed

On the

acid

indicate

of proteins

Conversely,

and activated

21,

in retinoic

results

common to a specific

retinoic

decreased

24 and 26 increased

animals.

RESEARCH

in For instance, as shown in -- situ. # 11, 12, and 13 is suppressed and that

was suppressed

animals

in

BIOPHYSICAL

formed

of

of proteins

synthesis in

17

AND

acid

synthesis

#

synthesis

was

by retinoic

3,

protein

retinoic

BIOCHEMICAL

1, 1988

The

rats

other

hand,

rats,

retinol

but

but

not

and retinoic

and distinct

on another

set

of proteins. Although on gene

it

appears

expression

(6),

synthesis If

this

described were

effects with

of

the

it

is

gene

sequential results

it

remains

compounds

mechanism

cascade-like

of about

Finally, activation

of of

the later

differentiation. affected

in

in

to

per

effects remain

whether and

it

during

acid

changes

in

first

of cytosolic

protein

mechanism.

the

fast

pleotropic interaction

an interaction

with

sequentially.

can

be

calculated

two hours proteins

is

after

would

If

such

from

the

refeeding

proceed

with

protein.

specific

genes gene,

of

retinoic

simultaneous

changes

indeed,

here

transglutaminase

rapid

whether

rapid

presented of

the

and

on transcriptional

a direct

turn

synthesis

results

report

only

1) that

retinol

Consequently, this

whether

retinol

or alternatively

(Table

repression

known

operative

18 seconds

the and

exception result

above

of

effects due

in

were

alterations

a cadency

are

trigger

action

to be elucidated

units,

would

presented

not

involve

many transcriptional

a single

primary

here case,

these

that

suggest thus

specific

far

represent and

rather

strongly

reported

(6),

a phenomenon retinoic

"early"

acid genes

that with

the

which on

is

a

cellular

suggested

to

be

to be characterized.

ACKN-S

This research was supported by NIH Grants grant from General Foods Corporation. The authors for technical assistance.

HD-07043 and HD-09195 and a thank L. Chytil and M. Hunt

RJPERENCES

1. 2. 3. 4. 5. 6. 7.

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BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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