Vol. 166, No. 3, 1990 February 14, 1990

BIOCHEMICAL

ACl’IVINA

S-PI-

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1479-1484

MEE(n?C-ONOFTHERAToo(1yTE INvrlRo

Masahiro ITOH' , Masao IGARASHI', Kiyohiko Moritoshi

YAMADA', Yoshihisa HASEXXWA'

SEKI', Yuzuru EttX2, and Hiroshiro

SHEAI

' Department of Obstetrics and Gynecology , GunmaUniversity Medicine, Maebashi, GUNM 371, Japan 2Central

Research laboratories,

Ajincmoto

School of

Co., Inc., Kawasaki

Japan

210,

Received December 25, 1989 SUMMARY:The effect of activin A on meiotic maturation was analyzed in oocytes from imnature rats treated with PMSG. Activin A, which was purified as the erythroid differentiation factor, accelmaturation of not only follicle-enclosed occytes and erated the oocyte-cumulus canplexes, but also denuded oocytes, as measured by an increase in the percentage of oocytes with germinal vesicle breakdown ( GVBD ). maturation was not accelwyte erated by activin A in the presence of the inhibitor of GLED such as cyclic-AMP. These results showed activin A is a potent in vitro stimulator of occyte maturation. 01990 Academic

Press,

Inc.

Meiosis

of mammalian oocyte

arrested

in

the

During puberty, increased

dictyate meiosis

IGF-I,

oocyte maturation. factor,

to

erythroid exactly strong (10-12).

stimulate

cccyte

same chemical

FSH releasing It

has

activity

such

that

been shown that

a

kind

of growth

But

homology

other

A (9),

same as in

activin

A

was reported

was demonstrated

was the

as

stimulate

On the

activin

the

to

to inhibin

as

birth.

factors

reported

maturation(g).

structure

after

maturation.

action

and is

of

inhibin,

factor(EDF)

life,

stimulation

grawth

cczyte

biological

differentiation the

the

and IGF-11(7) are recently,

fetal puberty

by

Recently

to inhibit

which has an opposite not

resumes

Quite

was found

in

stage until

gonadotropins(l-5). 'IGF-B(7),

M;F(6),

begins

chemical

hand,

to

have

and

a

activin

A

strucOOD6-291X/3/90 $1.50

1479

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in an-y form reserved.

Vol.

BIOCHEMICAL

166, No. 3, 1990

ture

exists

between activin

have

strong

FSH releasing

whether

activin

activity

or

A

AND BIOPHYSICAL

A

and IW-8

activity.

might

In

have

RESEARCH COMMUNICATIONS

and both this

study,

the

of

them

we tested

oocyte

maturing

not. MEX'HODS

The oocyte culture was performed according to the method Inmature female Wistar rats( 25 days old) of Feng et aL(7). were injected with PMSG ( 201U ) for 48 hours before removal of ovaries. To isolate the oocytes, follicles were punctured with a 26 gauge needle under a dissecting microthe follicular contents were expressed into the and scope, culture medium, HTF medium (13), containing 0.7% BSA Preovulatory cumulus-oocyte ccanplexes were isolated with a glass pipette, collected into the above medium, and kept on ice until the addition of hormones. To prepare denuded oocytes, the isolated cumulus-oocyte complexes were transferred to fresh medium, and cumulus cells were removed by passing the through a series of decreasing ccanplexes micropipettes of diameters. Each group containing denuded oocytes or cumulusoocyte complexes was incubated in 1 mlmadium with indicated hormones in a 60 x 15 mn dish with a center well(Falcon 3037). The cultures were incubated at 37’C in a 95% air - 5% humidified CO2 environment for the indicated times (1, 2, 3, and 4 hours). The oocyte-cumulus complexes were then placed on ice until the examination for meiotic maturation Follicle-enclosed oocytes were prepare excision of the by into fragments outer edge of the ovary containing about 10 follicles. They were incubated in the same mnner as cumulus-cocyte canplexes. After the indicated times, the follicles were ElTlOVed the ovarian from tissue and the oocytes were recovered for microscopic examination. Oocyte maturation was determined by Nomarski optical microscopy or phase contrast microscopy. Meiotic arrest was indicated by the presence of germinal vesicles (GV) and a nucleolus, while breakdown of these nuclear structures (GVBD) was an indication of the resumption of meiosis and oocyte maturation. Activin A ( erythroid differentiation factor (EDF) ) was purified from the conditioned medium of the humanmonocytic leukemia cell line THP-l(9) and dibutyryladenosine-cyclic monophosphate (dibutyrylcAMP) was purchased from Si gly chemical canpany. Stastical analysis was conducted with x test or paired t-test when comparing data for two groups. Duncan's multiple range test was used for analysis of differences between multiple groups. RESULTS The effects

of activin

in

denuded oocytes

At

1

hour

maturation. 61.6

+ 8.7

cultured

of incubation, But % of

A on

at

germinal

over 4 hours activin

2

hours

oocytes

with

of

A

did

are

breakdown(GVBD)

presented in

not

incubation,

100 rig/ml

1480

vesicle

activin

stimulate

Fig. 1. oocyte

GVBD occurred A,

in

77.2 + 11.0 %

vol.

No. 3, 1990

166,

BIOCHEMICAL

100

-.-

actwin

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

A 500 ng/mP

I 0

2

1 Incubation

3

Time

4

(hours)

FIG.l, Time course of activinA induced oocyte maturation. Denuded oocytes were incubated in the absence or presence of 100 rig/ml or 500 rig/ml activin A. Oocyte maturation, which was indicated by the percentage of GVBD, was measured. In this and the subsequent figures, total number of oocytes examined is indicated by the number in parentheses directly above or below symbols. Error bars represent +SEM.

with

500 rig/ml

The addition

(

rig/ml

of

However

at

groups

was

The

4

canplexes

Duncan's

100 rig/ml

% in and

maturation

range

test

significantly

increased

hours,

no

significant

difference

group. induced

denuded

A

also

A showed

observed

1.

of

significant

Effects

activin at

A 2

on

hours

follicle-enclosed

a

oocytes ).

at

At

3 hours,

GVBD

(p