BIOLOGY

OF REPRODUCTION

21, 1287-1293

Rat Oocyte

(1979)

Maturation: CARL

Effects

EKHOLM

and

Department

CLAES

Synthesis

Inhibitors

MAGNUSSON

of Physiology,

University S-400

of Protein

of Giiteb

33

org.

G$teborg,

Sweden

ABSTRACT Puromycin has been reported to block the meiosis of cultured mouse oocytes before extrusion of the first polar body, but to allow resumption of meiosis as revealed by germinal vesicle breakdown (GVB) (Schultz and Wassarman, 1977a). The conclusion from this was that GVB occurs independently of protein synthesis. In the present study rat oocytes were treated with puromycin during two successive culture periods. During the first period resumption of meiosis was prevented either by the concomitant presence of dibutyryl-cyclic AMP (dbcAMP) or by incubation of oocytes within their intact preovulatory follicles. Isolated oocytes, with or without cumulus cells, were then transferred to plain

medium

or medium

with

puromycin

alone

for

a second

culture

period.

Cumulus-surrounded oocytes matured spontaneously in culture. DbcAMP prevented GVB, if added within 1 h from the start of culture, whereas puromycin did not prevent GVB but blocked polar body formation, thus confirming earlier observations. When oocytes were first cultured for 0.5-4 h in the presence of both puromycin and dbcAMP and then cultured for 4 h with puromycin alone, GVB was prevented in a “time dependent” way: the longer the first culture period the lower the rate of GVB. The inhibition of GVB was, however, fully reversed when no drugs were present during the second culture. The block of GVB produced by puromycin was sustained even when the second culture was continued for 18 h. Cycloheximide, but not puromycin aminonucleoside (PAN), produced the same effects as puromycin. Similar results were obtained when denuded oocytes were used, suggesting that the effects of puromycin were directly on the oocyte and not mediated via the cumulus cells. When follicle-enclosed oocytes were incubated for 3 h with puromycin and the oocytes then isolated and cultured with puromycin, GVB was also inhibited. The rise in oxygen consumption seen in maturing oocytes could be prevented in oocytes maturing in medium containing puromycin. Also this effect of puromycin was reversible, indicating that puromycin did not inflict permanent damage on the oocyte. The interpretation of these data is that resumption of meiosis is dependent on some proteins with high turnover rates existing in the oocyte before the start of meiosis. Polar body formation might also be explained in a similar way.

Mammalian dictyate stage shortly

(oocyte preovulatory

ration”)

oocytes

1962). inhibited

of

peak by

preovulatory

it of

can

also

Meiosis

and

cultured

um

meiosis

in

is

a

et

is not

resumed

suitable

resumed

in of

to

(Tsafriri

follicles are cultured without when the oocytes are isolated

the

meiosis

al.,

hormone,

from

the

milieu

as but

follicles

hormone-free

medi-

(“spontaneous

matu-

vivo..

includes

also

specific

pattern

1287

unless

by

and

breakdown,

dissolution

the GVB),

of

nucleolus separation

chromosomes and extrusion body (PB). Normal maturation not

only

nuclear

cytoplasmic

of in

maturation

events

of

but

importance

subsequent (Thibault

cleavand

1973).

the

role

of

proteins

of

undergoing maturation during recent years.

September 11, 1979. July 3, 1979.

milieu,

a hormonal stimulus Meiotic maturation is

by

1976).

for normal fertilization and age, “cytoplasmic maturation”

Both

Received

vesicle

is

maturation

intrafollicular

altered

al.,

Edwards,

1935;

oocyte

characterized membrane

homologous the first polar

Gerard,

Accepted

is

et

of

Uil-

as long

Enzmann, that

the

by

(germinal

isolated

and

indicates

morphologically the nuclear

in

1972;

(Pincus This

(Tsafriri

the the

be initiated

gonadotropins

follicles

lensj#{246},1976).

in the prophase

Resumption of normally occurs as a consequence

and

addition

arrested meiotic

are first

the

after birth. maturation) follicle

LH-FSH vitro

INTRODUCTION

protein

synthesis

synthesized Thus,

and in

the

oocytes

have attracted interest several authors have

1288

EKHOLM

studied

the

acids

incorporation

into

and

Van

drugs

whether

normal

for

oocyte

1969).

Wassarman

various

drugs

specific arrested

protein

could

al.

concluded was not

that

oocyte

before

for

J agiello,

maturation

PB

at

protein

synthesis

GVB.

spontaneous

maturation

very

occurs

rapidly, the fact that puromycin did not affect GVB might be due to a slow onset of its effects. We have therefore used cAMP, which is a well known in

of

inhibitor

isolated

(Cho

resumption

et

al.,

Hillensj#{246}, 1977) and et al., 1978) oocytes. with

puromycin

ods, during inhibited (dbcAMP)

or

Rat

first

the

stances

puromycin

(Hillensj#{246}

oocytes

successive

of

which

were maturation

was

During

the

these

oocyte

Morphological

in

3-5/flask,

bicarbonate

Examination

The oocytes ference contrast

0.5

ml

(CaC12, C02,

buffer

of Oocytes

were examined by Nomarski intermicroscopy immediately after culture

and classified intact germinal

as: 1) immature (dictyate) oocytes vesicle and nucleolus; 2) oocytes vesicle breakdown (GVB); or 3) oocytes

germinal polar body less than excluded

Oxygen method tension cumulus

METHODS

(PB).

5% from

Degenerated

oocytes

of the examined the calculations.

with with with

were generally

oocytes

and

were

of Oxygen

Consumption

consumption of oocytes (3-4/measurewas determined by a spectrophotometric using hemoglobin as an indicator of oxygen (Hultborn, 1974; Magnusson et al., 1977). The cells were removed before respirometry.

Chemicals

under standard. conditions (lights on 0500-1900 h) were injected s.c. on Day 28 of life with 8 IU pregnant mares serum gonadotropin (Gestyl, Organon). This treatment leads to follicular development and an endogenous gonadotropin surge between 1500-1800 h on Day 30, with subsequent physiological ovulation of a normal number of ova between 0200-0500 h on Day 31 (Hillensj#{246}et al., 1974; Herlitz et al., 1976). Animals were killed between 0900-1300 hon Day 30 (before the endogenous gonadotropin surge) and the ovaries removed. Sprague-Dawley environmental

housed

rats

of Oocytes

Cumulus-enclosed incising the large

and

were

follicles

Follicles oocytes were considered

obtained

by

be preovulatory and releasing the cells into culture medium. In certain experiments oocytes were denuded by repeated sucking through mouth-operated micropipettes (Magnusson et al., 1977). This treatment resulted in complete detachment of the cumulus cells without grossly damaging the oocytes. Whole preovulatory

follicles

incubated,

1.25 mM), pH 7.5, equilibrated with 95% 02:5% with addition of 5.5 mM glucose and 10 mg/mI bovine serum albumin. After incubation, the oocytes were isolated from the follicles and cultured under the conditions described above for isolated oocytes.

ment)

Animals

Isolation

were Krebs-Ringer

Determination

circum-

GVB.

AND

10 i.tg/ml,

AMP

of

affect

MATERIALS

peri-

dibutyryl-cyclic

follicle. did

treated

culture

incubation

intact

both and

Magnusson

2

by by

within

1974;

follicle-enclosed

for

the either

of meiosis

of dbcAMP and puromycin was respectively, unless otherwise

concentration

1 mM and indicated. Follicles modified

and

formation)

Procedure

Isolated oocytes, cumulus-surrounded or denuded, were cultured, 20-50/dish, in 1 ml medium in organ culture dishes (Falcon plastics, 15 X 60 mm). The oocytes were cultured aseptically at 3 7.5#{176}C in humidified air (90% saturated with water) in a phosphate buffered medium containing 5.5 mM glucose, 25 mM DL-lactate, 0.25 mM pyruvate and 1 mg/mI bovine serum albumin (essentially fatty acid-free) and antibiotics (Magnusson et al., 1977).

The

that

that puromycin circular bivalent

concomitant

necessary

Since

is necessary showed

found in the

but

GVB

analyze

1968;

(1976)

block

1977).

to

synthesis

et

1976; Schultz

al.,

used

(Donahue,

stages. They the meiosis (after

stage

been

maturation

et

Warnes

have

during

Stein, 1977;

Blerkom,

1977b;

Wassarman,

and

patterns

MAGNUSSON

Culture

amino

proteins

and

(Golbus

and

Different

of

in protein

maturation

McGaughey

labeled

classes

changes

the

followed meiotic

of

different

AND

isolated

by

careful

‘-dibutyryl adenosine 3’, 5’cyclic mono(dbcAMP), puromycin dihydrochloride, puromycin aminonucleoside and cycloheximide were purchased from Sigma Lad, USA. All other chemicals were purchased from Sigma Ltd or Merck Co. N’

,O2

phosphate

Statistics Oxygen consumption number of samples). analysis of variance

is given

as mean

±

SEM

(n

Differences followed

to

microdissection.

=

were calculated with by Student-NewmanKeuls multiple range test (Woolf, 1968). P (0

50

puromycin

alone.

Isolated incubated

for

follicles. Intact 3 h in the absence

follicles

were

or presence

0-

of

0-

puromycin

(80

cumulus-enclosed follicles

pg/mI).

cultured

puromycin

present

during

was depressed present during

of GVB

periods, GVB).

it did

With

After

oocytes

and

without

was

4),

results

panel). Figure

s

the

by

first

alone (Fig.

When,

(1976).

inhibited

to

10

shown).

maturation

cumulus-surrounded

time

a longer

00

on

of puromycin

Denuded

of

reversible,

and 80 inhibiting

effective,

combination

before

fully

10

experiments

results

mycin

reversed effect

PB

al.

during

inhibitory

was

with

Since

other

good et

The

combined

not

was

0, 2, 4

18 h in puromycin

but

in

3). alone

required

oocytes

GVB

trans-

additional

was

oocytes

Wassarman

of

the

puromycin

although

is

(Fig.

dbcAMP

Also

and

an

meiosis

combination

(data the

h.

for

dbcAMP and

the

concentrations

was all

panel).

to

2

of

2 h with

puromycin

puromycin

resumed

(10

in

1, upper

within

dbcAMP

and

culture

maturation

analogue

or

h with

tg/ml.

Similar

aminonucleoside did not affect (Fig.

4

80

puromycin

of

period

GVB

maturation

presence h

in

in

puro-

maturation

2

tested

cycloheximide

instead

3).

ooof

when

Three

same

oocytes.

when

1,

presence

oocytes

1 j.ig/ml

was

isolated

first

and

the

while

.zg/ml

h culture on

for

and medium

dbcAMP were

(Figs.

6

cultured

plain

Culture

panel). were

effect,

oocytes

or

obtained.

by

dbcAMP to

effect

were

Puromycin were

with

allowed

of the

upper

dbcAMP:

inhibition

concomitant

the

40%

puromycin

pg/ml

the

medium

continued

was

1,

of

Cumulus-surrounded

by

only

(Fig.

when

6 h in the meiosis, but

during

alone,

addition

The

and

ferred

results

of the

Oocytes or

1289

similar

of GVB

dbcAMP

which

for

inhibited

culture

Reversibility Inhibition

present

MATURATION

respectively),

4 h

for

the

OOCYTE

the

1977).

was

RAT

‘\‘8

to a very

drug

incubation.

oocytes.

dbcAMP

of

GVB

cultured resumed

was

al.,

cultured

When

is

Synthesis

on

Isolated mycin

et

meiosis

plain

Protein

Inhibition

cytes

4

reversed

transferred of

most

oocytes

culture.

1

GVB

begins h

resumed the

Within of

10

when

ON

delayed 15, 30 or 60 mm, meiosis in 38, 54 and 82%, respectively, at

of

Effect

extent

(Magnusson

of

dbcAMP was was resumed

meiosis.

the

within

Cumulus-surrounded with

PUROMYCIN

h. PB formation

and

have

oocytes

resume

GVB

within

after

OF

were for

bh

culture

to 95% had (80 and 10 pg/ml,

,ned,6,,,

2-6h

FIG.

1. Effect

C

of protein

k,

synthesis

Pr

C

inhibition on GVB in isolated oocytes. Cumulus-surrounded oocytes (upper panel) or denuded oocytes (lower panel) were cultured for 2 successive periods, for 2 h and 4 h, in plain medium or in the presence of dbcAMP (1 mM) and/or puromycin (10 Mg/mI) or PAN (80 g/ml) as indicated. The rate of GVB was determined after the 4 h culture period. (Number of oocytes indicated within the bars.)

1290

AND

EKHOLM

MAGNUSSON

were

oocytes

100-

thereafter increase was

6-8 seen

was

present

the

> 050

cultured

for

rate

the

of respriation

entire was

whether effect

That

the

the

incu

time

-

-

-

0

0.5

I

4

4

4

I

tlmescub.2

FIG. 2. Time-course of the dbcAMP and puromycin on GVB. oocytes were cultured for 0-4 dbcAMP (1 mM) and puromycin ferred to medium containing pg/mi)

for

additional

determined

was

(Number

[Ilci

puromycin

-

4 4h

combined

h

effect

of

Cumulus-surrounded

h in the presence of (10 pg/mI) and transonly puromycin (10

4 h culture.

after oocytes

of

2

4

The

rate

of

consumption noncultured

cantly

the

alone,

reduced.

rate

When

of

the

GVB

transferred to plain medium, the and PB formation was approximately as for

oocytes

cultured

respiration

period,

significantly had

GVB

when oocytes of both dbcAMP transferred

and

rate

not

differ after

and

was further

were and plain

to

period. oxygen

from that 8 h culture

present

et al.

Cho

study

(1974) of

confirms

that

the

culture

dbcAMP

of

inhibits

from

data

oocytes

in the

resumption

of

signifi-

dbcAMP

rate

of GVB the same in

was

was no

thus

of be

effects

delayed

longer

Failure

plain

of the

increased.

instead

continuously

or

revers-

was

DISCUSSION

The

as

attributed

to

act

to

as the

some

time

same

slow

To

synthesis.

before

mm,

15

inhibit

a too

to

protein

to

little

inhibited puromycin

on

mycin by

instead

were

medium. Oxygen

puromycin

culture

meiosis but only if dbcAMP is present from start of the culture. When the addition

was

oocytes

h

2

did oocytes

presence puromycin

and a similar

medium for another 4-8 h culture After 4 h culture in plain medium, their

GVB

the second culture period. indicated within the bars.)

for

dbcAMP

oocytes puromycin

of

ible was demonstrated cultured in the presence

0-

2 h in

plain medium, (Table 1). When

during

decreased not.

for

h in

the of GVB

extent.

GVB

might

onset

of

its

allow purothe onset of

Consumption

Cultured

Oocytes

was

It

maturing

earlier in

respiration

shown

plain

that

medium

(Magnusson

cultured

oocytes

increase

their

al.,

1977).

et

rate

R of

GVB PB

When 00-

-

-

-

00

LI

acAMP

C

0

4AUP

C

m >

,P1,

-

o5050

036 -

0 time

kicubl

timerncub2

FIG.

-

49

------

61

6

42

2

2

2

h

0

2

4

6

h

of the

effect

0-

medum

2-20h

h

92

PAN

Pur

C

PAN

Ri

of puromycin

on for

GVB. Cumulus-surrounded oocytes were cultured 2 h in medium containing dbcAMP (1 mM) without (open bars) or with (hatched bars) puromycin

(10 Mg/mI) and then either transferred to plain medium for an additional 2-6h culture period or examined directly for GVB. After the second culture period, the rate of GVB was determined. (Number of oocytes indicated within the bars.)

64,

dbcAP #{149}Pur

-

2

3. Reversibility

2

medssn

60

C

FIG.

Pur

dbcAMP ‘Pur

C

4. Effect of protein synthesis inhibition on GVB and PB formation. Cumulus-surrounded oocytes were cultured for 2 h and then transferred to new media for an additional 18 h culture period. Culture was carried out in medium alone or in medium containing PAN (80 pg/rnl) or puromycin (10 pg/ml) alone or in combination with dbcAMP (1 mM) as indicated. After culture oocytes were examined for GVB (total bar height) or PB (hatched portion of bars). (Number of oocytes indicated within the bars.)

EFFECTS

TABLE

First

1. Oxygen

OF PUROMYCIN

consumption

of isolated

culture

oocytes

Second

Experiment I Not cultured

Puromycin dbcAMP

Not

ON RAT

after

OOCYTE

MATURATION

1291

culture.a

Maturation stage of oocytes taken for respirometry

Oxygen consumption (nl/h/oocyte)

culture

0.123

±

0.085

±

8 h

0.091

±

8 h

0.165

Dictyate

0.011 0.004

(4) (3)

GVB

0.006 0.007

(3)8

Dictyate

±

(5)

GVB

0.133

±

0.008

(9)

Dictyate

4 h

0.154

±

0.010

(7)

8 h 6 h

0.182 0.179

+

0.005

(11)8*

cultured

2 h

Puromycin

8 h

2 h

Puromycin

2 h

Plain medium

+

puromycin

dbcAMP Experiment 2 Not cultured dbcAMP puromycin

Not cultured

or PB

+

dbcAMP + puromycin dbcAMP

2 h

Plain

2 h 2 h

Plain medium Plain medium

aCumulussurrounded

oocytes

medium

were

cultured

GVB GVB

(9)

± 0.006

2 h in medium

for

GVB

NS

containing

dbcAMP

(1 mM)

or PB

and/or

puro-

mycin (10 pg/mI) and then transferred to new media, either plain or containing puromycin (10 pg/mI) for an additional 4-8 h culture period. After the second culture period oocytes were denuded and oxygen consumption recorded. Oocytes taken for respirometry were selected to get uniform maturation in each group. As control, oxygen consumption of noncultured, denuded oocytes was measured. Oxygen consumption is given as the mean ± SEM (number of recordings in parentheses). P