Undegraded vitellogenin polysomes from female insect fat bodies

BIOCHEMICAL

Vol. 78, No. 2, 1977

UNDEGRADED VITELLOGENIN

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

POLYSOMES FROM FEMALE INSECT FAT BODIES Franz

Engelmann

Department of Biology University of California, Los Angeles, California 90024

Received

August

12,1977

SUMMARY. Fat body cells of vitellogenic females of the cockroach Leucophaea maderae contain a prominent population of large polysomes of approximately 35-40 ribosomes whereas fat bodies of non-vitellogenic females or males of any age do not have these polysomes. Anti-vitellogenin recognizes newly synthesized nascent vitellogenin associated with these large polysomes. Adenosine labelled RNA is likewise precipitated by anti-vitellogenin primarily in the region of this class of polysomes. It is concluded that the class of large polysomes represents the vitellogenin polysomes. Vitellogenins proteins ing

in the

via

fat

high

synthesized

the yolk

found

that

about

protein

vitellogenin (2)

and amphibians

of insect

species

(4,s).

In the

endoplasmic This

reticulum

mechanism

proteins

cells

of the

process of the

an identifiable

and secreted is

implies

that

insect

of vitellogenin

protein

Leucophaea

on the

However,

synthesis.

of a hormone

0 I977 by Academic Press, Inc. of reproduction in any form reserved.

(6,~)

that

directs

differentiated

into

the

other

cisternae.

exportable

of the

identification

fat

body

of specific

reported. for

of particular

The

an understanding interest

the massive tissue.

surfaced

synthesizing

reticulum

is

and the

on rough

for

is essential It

i.e.

maderae

has not been

polysomes

It

in the majority

polysomes

to date

vitellogenin

in a fully

shown

endoplasmic

con-

hormone

synthesized

specific

proteins

by hormones,

discharge

to that

at

from vitellogenin.

or juvenile

are

the

and birds

directed

by vectorial

similar

of such specific

involvement

(3)

cockroach

females.

synthesizing

is

grow-

are produced

of the yolk

derived

vitellogenins

must be located

characterization

Copyright All rights

This

of vitellogenic

polysomes

(8)

of secretion

(9,lO).

vitellogenin

cause

migratoria

is

synthesis

in birds

Locusta

90 percent

yolk

up by the

vitellogenins

of amphibians

which

estrogens

locust

livers

predominant

and taken

egg growth

or the

In insects

rates.

to the

extraovarially

During

of insects

of vitellin,

has been

as the precursors

pinocytosis.

bodies

extremely sist

They are

(1).

oocytes

are defined

be-

production

I have

of

now obtained

641 ISSN

0006--7Vl.Y

Vol. 78, No. 2, 1977

evidence sizing

for

BIOCHEMICAL

the existance

polysomes

in the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of such a specific fat

bodies

of the

class

female

of vitellogenin

cockroach

synthe-

Leucophaea.

MATERIAL AND METHODS Colonies of the cockroach Leucophaea maderae are maintained at 26Oc and approximately 75% relative humidity. Newly emerged females were collected from these colonies and set aside for later use. For polysome preparations 70-80 mg of fat bodies were homogenized in Tris-HClbuffer (2OOmM) at pH 9.0 containing 200mM sucrose, 4OOmM KCl, 35mM MgC12, 1% Triton X-100, and 25mM EGTA (ethyleneglycol-bis (S-aminoethyl ether) N,N'-tetraacetic acid) (11). Following two centrifugations at 10,000 g for 10 minutes each, the supernates were directly layered onto sucrose gradients of 15 to 60 percent. The gradients were made in Tris-HCl buffer (4GmM) at pH 9.0 and contained 200mM KCl, 35ti MgC12, and 5mM EGTA. After three hours of centrifugation in a Sk? 27.1 rotor at 76,000 g the gradients were emptied while absorbancy was monitored at 254 nm using an ISCO UV analyzer. Two ml fractions were collected. Aliquots of each of the fractions were diluted with buffer before anti-vitellogenin or antiserum to male hemolymph were added. After one hour incubation at 37'C, carrier protein (diluted female hemolymph) was added and then refrigerated over night. The precipitates were centrifuged down, washed once in buffer and trichloroacetic acid, and then redissolved in NaOH for counting. RESULTS In a typical

absorbancy

females

a prominent

portion

of the

characteristic vitellogenin

with

peak of

sucrose

density

for

bodies

fat

synthesis

destroyed

(egg

by ribonuclease

EDTA (1OmM).

homogenates

the

fat

of polysomes

nndegraded

from size

bodies

appearance

(Fig.

females

unique

I suspected

that

in vitellogenin

synthesis.

To show this

were

collected

mg of tissue aliquots

24 hours used for

from each

to male hemolymph, vitellogenin

after

density were

and the precipitates

recognized

antigens

bodies

with

642

intense

most

g supernate

observed

is

in

of any age.

correlated

of polysomes

with involved

from vitellogenic

of 40 uCi of %- adenosine analysis.

After

as before

associated

with

females and 80

fractionation,

anti-vitellogenin

processed

primarily

are

or males

peak

a class

fat

of

of the 10,000

females

gradient

treated

polysomes

peak was never

contained

an injection

sucrose

fraction

it

lower

They can be completely

absorbancy

vitellogenesis,

seen in the

the period

or treatment absorbancy

from vitellogenic

These

during

of non-vitellogenic of the

is

1).

of 2 to 4 mm).

prominent

obtained

polysomes

gradient

(1 ug/ml),

This

from

Since

profile

or antiserum (12). the

class

Antiof

BIOCHEMICAL

Vol. 78, No. 2, 1977

I

vdellOgenrc

t

vrtcbogenic

E ::

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

female

fat

female

lot body

EDDrA

I

body

mole

lot

body

I IUmM)

iz E

/ 5

10

5

IS Fract!on

15

10

Number

of fat bodies from vitellogenic females and males. Fig. 1. Polysome profiles In all cases 80 mg of fresh fat body tissues were used. Fat bodies were homogenized in a medium containing EGTA. After centrifugation at 10,000 g disodium EDTA or ribonuclease (bovine pancreas) was added to the supernates and then layered directly onto the sucrose gradients.

labeled

large

lo-15

polysomes

percent

fact

is the

carrier

of the result

to the

ribosomal

subunits

cipitated

with

polysomal

degradation

large size

incubation

with

the

polypeptides.

between density

ribosome

not

precipitate This

due to the

media.

In fractions

containing

a certain

amount

may indicate

provide

of vitellogenin

was estimated monomeric

a certain

degree

gradient.

643

evidence

and distance

of the

of large pre-

of

products

polysomes.

by extrapolation units

the

was also

strong

arti-

addition

of label

the degradation

data

more than

vitellogenin.

of counts

These

of 35 to 40 ribosomes

anti

preparation,

the class

did

trapping

This

during

represent

antisera

precipitable

anti-vitellogenin.

polysomes

sucrose

Male

and the monosome

nascent

relationship the

label

2).

of non-specific

antigens

carrying

(Fig.

that

still the

A polysome log-linear

of migration

in

Vol. 78, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

e-i-.

*

2

*.

*

I

6

4

Polysome profiles of fat bodies from vitellogenic females and males. ~~'a%nals had been injected with 40 yCi of [2,8-3H]-adenosine (spec. act. 30.4 Ci/mmol, NEN) 24 hours prior to collecting the tissues. After sucrose density gradient centrifugation and fractionation aliquots of the fractions were treated with anti-vitellogenin or antiserum to male hemolymph and the precipitates processed as before (12). Fig. 3. Polysome profile of fat bodies from vitellogenic females which had been injected with 1 uCi of [14C(U)]-leucine (spec. act. 314 mCi/mmol, NE%) two hours prior to collection of the tissues. The labelled vitellogenin of each fraction was precipitated by treatment with anti-vitellogenin. Since pelleting of the polysomes prior to sucrose density gradient centrifugation had to be avoided, large amounts of vitellogenin freed during homogenization and dissolution of the microsomal vesicles now float on top of the gradient.

Fat bodies above did

prepared

not yield

genin

(Fig.

label

in the top

male

fat

2).

bodies

circumstances 14 C-Leucine found

almost

the

from males very

large

Anti-vitellogenin of the are

the

polysomes

same procedure

this

by anti-vitello-

however,

can be considered

to produce

as described

precipitable

precipitated,

gradient;

not known

with

vitellogenin

a certain an artifact,

--in vivo

under

amount since any

(12). labeled

polypeptides

exclusively

associated

precipitable with

644

the

by anti-vitellogenin class

of large

polysomes.

were The

of

BIOCHEMICAL

Vol. 78, No. 2, 1977

radioprofile area

followed

(Fig.

tated

3).

Thus,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

very

closely

both

newly

synthesized

nearly

entirely

by anti-vitellogenin

that

of the

absorbancy

at 254 nm in this

RNA and protein in the

region

can be precipi-

of the

large

polysomes. During apparent

the many attempts

that

to or after degraded

pelleting

of microsomes

detergent

treatment

or the

10,000

polysomes.

as nuclease

The unique suggests

that

the

large

very

about

in producing

most

of the

of the

is vitellogenin

(Table

hemolymph

is vitellogenin,

the

proteins.

total

released before

profile

proteins 1).

Oocytes

to a low

87 percent

of the it

phase

intact these (0.05%)

in the

absence

of

synthesis

up the

the

about

on

oocytes hemolymph

proteins

newly

and release

occurs

of the

labeled

completely

females

into

makes up only take

and ceases

of the

tissues

and released

preferentially

level

were

destroyed

media

of these

the maximum growth

Vitellogenin

polysomes

of vitellogenic

activity

synthesized

While

prior

polysomes.

bodies

quantitatively

vitellogenin. ovulation

fat

became

g either

Freezing EGTA also

it

or diethylpyrocarbonate

the specific from

During

large

and gradient

translational

polysomes.

87 percent

or without

in homogenization

polysome

the

(1 mg/ml)

polysomes

at 100,000

in the media.

with

The use of heparin

EGTA was ineffective

vitellogenin

was detrimental:

g supernate

inhibitors

the

or polysomes

even when EGTA was contained

tissues large

to identify

of the

5 percent

synthesized

declines

of and

shortly

at ovulation.

DISCUSSION The reliable by the

use of the

prior

to gradient

equally were

This

Ca chelator

successful. of

sensitivity

EGTA (11).

This

sensitive of the

points

published

to the

polysomes

of the

fact

that

the

vitellogenin during

to degradation

645

high

was made possible

of polysomal

pelleting

tissues

however,

procedures

RNases and that to degradation

polysomes

Omission

or freezing

None of the

Ca dependent

extremely

of vitellogenin

centrifugation

important.

amounts are

identification

was,

on polysome fat

bodies polysomes

isolation

contain

large

of insects

speed

centrifugation.

may argue

against

the

notion

BIOCHEMICAL

Vol. 78, No. 2, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1. Incorporation

of l4 C-leucine

rates

Oocyte

size

into

Number

mm

serum proteins

Total

of vitellogenic

serum proteins

females.

Vitellogenin

of spec.

CPrn/ l 1-11

act.

spec.

cpm/ l Ul

act.

animals cpmlug 2.3

- 4.0 5.0a

protein

6

359 + 91"

9.7 + 2.4

322 + 94

3

49 + 11

2.7 + 1.6

34 + 19

14 C-leucine injected with 1 uCi Proteins of aliquots of the sera were precipitated trichloroacetic acid or anti-vitellogenin and the previously described (12). The amount of vitellogenin determined by rocket immunoelectrophoresis. This enough to detect vitellogenin quantitatively in a from a vitellogenic female. * Mean -+ S.E. "Fully grown oocytes. Animals

bThis were

3 of the

'No determinations

of the amounts

that

peak in the

prominent

represents caused

an aggregation

male tissues

or those

yield

this

size

tions

(1OmM) did Recently,

identified

large

appears product

lower

portion

media

is

of polysomes,

and on the affect

vitellogenin

polysomes

males

case the

the

size

to be in the of Leucophaea

vitellogenin

of the order

primary of 200,000

vitellogenin

646

density

since

C

samples were

made.

gradient

of polysomes on the one hand,

treated

identically did ++ concentralower Mg

other, absorbancy

of 30-40

by immunoprecipitation contained

sucrose

Aggregation

females

--

other samples

unlikely,

substantially

the

in these

of the

polysomes.

of non-vitellogenic

liver

Xenopus

In either

polysomes translation

not

in avian

estrogenized (14).

class

because

of vitellogenin

of small

35 1124Mg++ in the

by the

samples,

232 + 74b

2 hr prior to bleeding. with either 10 percent precipitates processed as in the samples was technique is sensitive sample of 0.1 ul of serum

were

figure is based on only accidentally lost.

the

cwhg protein

profiles.

ribosomes

(13).

Also,

polysomes translation daltons. polysomes

have been liver

of

of 30 ribosomes product

of these

The primary is

not known

for

not

Vol. 78, No. 2, 1977

BIOCHEMICAL

certain,

the polysome

but

dissimilar

considering

to that

The unique

known

for

polysome

females

points

to the

at this

time.

It

is

Locusta

profile

fact

size (8),

of the

that

a ready

AND BIOPHYSICAL

this

source

one may predict i.e.

fat

the

that

approximately

bodies

tissue for

RESEARCH COMMUNICATIONS

it

is

250,000

not too daltons.

of reproductively

active

is making

primarily

vitellogenin

isolation

of the vitellogenin

messenger. For any further biosynthesis

in the

genin

mRNA is

genin

polysomes

crucial.

understanding fat

bodies With

of the of an insect

the

we are now in the

successful position

detailed the

mechanisms availability

of the

identification to perform

of vitellogenin

of the the

critical

vitello-

vitelloexperi-

ments. ACKNOWLEDGEMENTS The research reported and the National Institutes and J. Lengyel for critically suggestions. I also thank

here was supported by grants from NSF (GB 14965) of Health (AI 12878). I thank Drs. R. Goldberg reading the typescript and making many helpful Mr. S. Oda for technical assistance. REFERENCES

1.

Pan, M.L., Bell, W.J., and Telfer, W.H. (1969) Science 165, 39x-394. Gruber, M., Bos, E.S., and Ab, G. (1976) Mol. Cell Endocrinol. 5, 41-50. Tata, J.R. (1976) cell 9, l-14. 43: Engelmann, F. (1970) The Physiology of Insect Reproduction, Pergamon Press, Oxford. in Biochemical Actions of Hormones, 2, 385-490. Ed. 5. Wyatt, G.R. (1972) Litwack, G. Academic Press New York and London. 6. Engelmann, F. (1974) ARI. ZOOI. 14, 1195-1206. F., and Barajas, L. (1975) Exp. Cell Res. 92, 102-110. 7. Engelmann, a. Chen, T.T., Couble, P., DeLucca, F.L., and Wyatt, G.R. (1976) in The Juvenile Hormones, pp. 505-529. Ed. Gilbert, L.I. Plenum Press, New York and London. 9. Tata, J.R. (1973) Karolinska Symp. Res. Meth. Reproduct. Endocrinol. 6, 192-224. 10. Adelman, M.R., Sabatini, D.D., and Blobel, G. (1973) J. Cell Biol. 56, 206-229. 11. Jackson, A.O., and Larkins, B.A. (1976) Plant Physiol. 57, 5-10. 12. Engelmann, F. (1971) Archs. Biochem. Biophys. 145, 439-447. M., and Ab., G. (1976) Biochim. Biophys. Acta 435, 13. Roskam, W.G., Gruber,

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647

Henshaw,

E.C.,

and Tata,

J.R.