J. Embryol. exp. Morph. 73, 317-338, 1983Printed in Great Britain © The Company of Biologists Limited 1983

Time-dependent effects of a-amanitin on nuclearmaturation and protein synthesis in mammalian

oocytes

By J. C. OSBORN1 AND R. M. MOOR1

From the A.R. C. Institute of Animal Physiology, Cambridge

SUMMARY

The addition of a-amanitin to extrafollicular, cumulus-enclosed ovine oocytes at explanta-tion inhibits meiotic maturation and prevents many of the changes in protein synthesis thatnormally accompany maturation. By contrast, these inhibitory effects are considerablyreduced by either delaying the addition of the drug for 1-4 h or by denuding the oocytes of allassociated cumulus cells at the onset of culture. The observations that the inhibitory effect ofcordycepin on nuclear maturation is also time-dependent and cumulus-cell-dependent andthat the oocyte is susceptible to cordycepin for longer than its sensitivity to a-amanitin areconsistent with the differential effects of these drugs on RNA synthesis.

It is concluded that a transcriptional event at the onset of maturation is essential for theinitiation of those changes in protein synthesis required for the regulation of nuclear andcytoplasmic maturation. It is uncertain, however, whether this transcriptional event occurswithin the cumulus cells or within the oocyte.

INTRODUCTION

Although most of the RNA present in mammalian oocytes is synthesized andaccumulated during the period of oocyte growth (Bachvarova, 1974; Jahn, Baran& Bachvarova, 1976; Bachvarova & DeLeon, 1980; Sternlicht & Schultz, 1981;Piko & Clegg, 1982), it is clear that RNA synthesis continues at a low level towithin 1 h of germinal vesicle breakdown (GVBD) and that some of the newlysynthesized RNA is released into the cytoplasm before GVBD (Bloom & Muk-herjee, 1972; Rodman & Bachvarova, 1976; Wassarman & Letourneau, 1976a;Wolgemuth & Jagiello, 1979). Furthermore, there is evidence that poly(A)-containing RNA synthesis continues in fully grown oocytes (Brower, Gizang,Boreen & Schultz, 1981; Piko & Clegg, 1982).

The inhibition of meiosis in oocytes by actinomycin D (Donahue, 1968; Bloom& Mukherjee, 1972) suggests that transcription may be necessary for the com-pletion of the first meiotic division. However, other results show that meiosis isnot inhibited by actinomycin D when used at low concentrations (Jagiello, 1969;Golbus & Stein, 1976; Crozet & Szollosi, 1980) but that high concentrations

1 Authors' address: Institute of Animal Physiology, 307 Huntingdon Road, CambridgeCB3 OJQ, U.K.

EMB73

318 J. C. OSBORN AND R. M. MOOR

result in chromosomal abnormalities (Jagiello, 1969; Alexandre & Gerin, 1977).Since actinomycin D is not a specific inhibitor of messenger RNA (mRNA) atlow concentrations (Manes, 1973) and at higher concentrations has deleteriousside effects on protein synthesis, respiration and glycolysis (Honig & Rabinovitz,1965; Laszlo, Miller, McCarthy & Hochstein, 1966), the suppression of meioticmaturation by actinomycin D has been regarded with some caution. A morespecific inhibitor of the RNA polymerase involved in the synthesis of mRNA,RNA polymerase II, is a-amanitin (Lindell et al. 1970; Sekeris & Schmid, 1972;Tata, Hamilton & Shields, 1972; Weinman & Roeder, 1974). This particulardrug has been shown to be an efficient inhibitor of development in preimplanta-tion rabbit (Van Blerkom, 1977) and mouse embryos (Golbus, Calarco & Ep-stein, 1973; Warner & Versteegh, 1974; Levey, Troike & Brinster, 1977;Braude, 1979a,6) at concentrations which completely inhibit RNA polymeraseII activity in vitro (Versteegh, Hearn & Warner, 1975). We have therefore madeuse of this selective action of a-amanitin to determine whether new mRNAsynthesis is required for the initiation of either nuclear or cytoplasmic eventsduring the maturation of mammalian oocytes.

MATERIALS AND METHODS

Tissue preparation and culture methods

Ovaries were obtained from sheep injected on day 10-12 of the oestrous cyclewith 1250 i.u. of pregnant mare serum gonadotrophin and slaughtered 40 h later.Intact, non-atretic follicles were dissected from the ovaries at room temperatureand opened to remove the entire cumulus-oocyte complex. Two types of culturewere carried out: (i) the intact cumulus-oocyte complex was cultured (cumulus-enclosed oocytes) or (ii) the oocyte was cultured after removal of the cumuluscells with fine pipettes (denuded oocytes). Cumulus-enclosed and denudedoocytes were cultured at 37 °C in media containing 10 /ig ml"1 NIH-LH-S18 usingthe conditions and culture medium described by Crosby, Osborn & Moor (1981).For cultures with a-amanitin and cordycepin, cumulus-enclosed and denudedoocytes were divided into groups and exposed to a-amanitin (Boeringher-Mannheim, 10/igml"1) or cordycepin (Sigma, SOjUgml"1) at selected times afterremoval from the follicle (Figs 1A and B). In those groups of oocytes culturedwith a-amanitin or cordycepin from explantation (oh) all preparative procedureswere carried out in media containing the appropriate inhibitor. After 18 to 24 hculture, cumulus-enclosed and denuded oocytes were either examined as wholemounts after staining with lacmoid or radiolabelled with [35S]methionine forone- and two-dimensional gel electrophoresis.

Radiolabelling of oocytes

Groups of six to ten denuded or cumulus-enclosed oocytes were labelled at37 °C for 3h in 50 \x\ of incubation medium (Moor, Smith & Dawson, 1980)

RNA inhibitors and oocyte maturation 319containing either 500/iCi or lmCi/ml [35S]methionine (Specific activity >lOOOCi/mmol, Radiochemical Centre, Amersham). After incubation, groupsof denuded and cumulus-enclosed oocytes were washed once in incubationmedium and the latter were denuded of cumulus cells. Denuded oocytes werethen briefly washed in lOmM-Tris-HCl, pH7-4, collected in a small volume ofTris buffer (<5/A), lyophylized and frozen at -70 °C until required forelectrophoresis.

Electrophoretic analysis of oocyte proteins

Labelled oocyte polypeptides were analysed in one dimension as described byMoor, Osborn, Cran & Walters (1981), or in two dimensions essentially accord-ing to O'Farrell (1975) and O'Farrell, Goodman & O'Farrell (1977).

For one-dimensional analysis, groups of oocytes were lyzed in 25-30/il ofsample buffer (O'Farrell, 1975) and a 5/il aliquot used for determining incor-poration of radioactivity into TCA-precipitable material. Equal numbers ofTCA-precipitable counts were applied to a 8-15 % linear gradient SDSpolyacrylamide slab gel and the polypeptides separated for 3h at a constantcurrent of 20 mA per gel. Labelled proteins were visualized by fluorography(Bonner & Laskey, 1974) using preflashed' Kodak X-Omat film at -70 °C,(Laskey & Mills, 1975). Molecular weight determinations were made using a[14C] methylated protein mixture (relative molecular mass, Mr range 14-3 x 103

to 200 x 103; Radiochemical Centre, Amersham) as standards. Microden-sitometer scans were made of each fluorogram and a quantitative and statisticalanalysis of the changes in protein synthesis carried out as described by Moor etal. (1981).

For two-dimensional (2D) analysis of acidic and basic polypeptides, groups ofoocytes were placed in 20/ 1 of lysis buffer containing 9-5M-urea, 2% w/vNonidet P40 (Sigma), 5% mercaptoethanol and 2% ampholines (1-6% pHrange 5-7 and 0-4% pH range 3-5-10; LKB). After freezing and thawing thesamples twice, duplicate 1 jwl aliquots were used to determine the TCA-precipitable counts. Samples containing 100 000 c.p.m. in 15 /il were applied to4 % polyacrylamide gels consisting of 9-5 M-urea and 2 % Nonidet P40 with 2.8 %ampholines (2-4 % pH range 5-7 and 0-4 % pH range 3-5-10) for isoelectricfocussing (IEF) and 2 % ampholines (1 % pH range 7-9 and 1 % pH range 8-9-5)for non-equilibrium pH gradient electrophoresis (NEPHGE). After electro-phoresis at 400V for 18 h (IEF) or 4|h at 400V (NEPHGE) the gels wereequilibrated for 20 min in sample buffer before loading onto 15 % SDSpolyacrylamide slab gels. After electrophoresis, the gels were processed forfluorography and exposed to preflashed Kodak X-Omat H film for 3 days. Themolecular weights of unknown proteins in the second dimension gels were deter-mined by comparison with concurrently electrophoresed 14C-marker proteins(see above) added to the agarose bed upon which the IEF or NEPHGE gel wasplaced.

320 J. C. OSBORN AND R. M. MOOR

Uridine uptake and incorporation

Groups of cumulus-enclosed oocytes were labelled for 4 h in 50 [A incubationmedium containing 100/iCi [5,6-3H] uridine/ml (specific activity 40Ci/mmol;Radiochemical Centre, Amersham) in the presence or absence of lOz/gml"1

a-amanitin. After incubation, the oocytes were denuded, washed once in Trisbuffer and disrupted in 30 /il SDS sample buffer. Duplicate 2-5 /A aliquots of eachsample were used to determine total counts and the remainder of each sampleused for TCA-precipitable counts as described by Braude (1979a).

RESULTS

a-amanitin and nuclear maturation

The effect of a-amanitin on the resumption of meiosis was examined in 288oocytes cultured in a-amanitin at various times after explantation. Fig. 1A showsthat the presence of 10 \xg ml"1 of a-amanitin throughout culture reduced to 29 %the proportion of cumulus-enclosed oocytes in which GVBD and the formationof a metaphase plate had occurred. By contrast, the inhibitory effect ofa-amanitin on meiotic maturation was greatly decreased by delaying the additionof the inhibitor to cumulus-enclosed oocytes or by culturing the oocytes in theabsence of cumulus cells. Thus, when a-amanitin was added at either 1 h or 2hafter explantation, 60 % and 83 % respectively of cumulus-enclosed oocytesunderwent GVBD, while 73 % of denuded oocytes, cultured from explantationwith a-amanitin, showed normal metaphase plates. These results demonstratethat the maintenance of oocyte-cumulus cell contact is necessary for the inhibit-ory action of a-amanitin on nuclear maturation, but that the cumulus-enclosedoocyte is only susceptible to the inhibitor for a short period after the initiationof meiosis.

To confirm the specificity of action of a-amanitin, we have used a secondinhibitor, cordycepin, which blocks the post-transcriptional adenylation ofnuclear RNAs (Darnell, Philipson, Wall & Adesnik, 1971; Penman, Rosbash &Penman, 1970). Fig. IB shows that the effects of cordycepin on nuclear matura-tion are very similar to those obtained with a-amanitin being both time depen-dent and cumulus-cell dependent. In addition, the finding that the cumulus-enclosed oocyte is susceptible to cordycepin for longer than its sensitivity toa-amanitin is consistent with the differential effects of these drugs on the tran-scription and processing of RNA.

a-amanitin and protein synthesis

The results presented in Table 1 show that the incorporation of labelledmethionine into TCA-insoluble material in cumulus-enclosed and denudedoocytes is unaffected by a-amanitin. By contrast, the presence of a-amanitin

RNA inhibitors and oocyte maturation 321

100

80

60

40

20

(96)

(55)

GV

Metaphase

(42)

(20)

(30)

m

(45)

%100

80

60

40

20

Untreated

(38)

Oh

(40)

l h 2h 6h Denuded, Oh

Onset of a-amanitin treatment

GV

Metaphase (15)

(11)

(12)

(15)

Untreated Oh 4h 5h 6h Denuded, Oh

Onset of cordycepin treatment

Fig. 1. Nuclear development of cumulus-enclosed denuded oocytes examined 18 hafter (A) culture with lOjUgml"1 a-amanitin from 0,1, 2 or 6 h after explantation or(B) after culture with 50/^gmP1 cordycepin from 0, 4, 5 or 6h after explantation.Illustrated are the percentage of oocytes at the germinal vesicle (GV) and metaphasestages of development. Figures in parentheses indicate number of oocytes examined.

OJ

Tab

le 1

. In

corp

orat

ion

of [

35S

] m

ethi

onin

e in

to c

umul

us-e

nclo

sed

and

denu

ded

oocy

tes

afte

r 18

h c

ultu

re i

n th

e pr

esen

ce a

ndab

senc

e of

a-a

man

itin

(1

0 p.

gml~

l ).

A a

nd B

rep

rese

nt t

he l

evel

s of

inc

orpo

rati

on

calc

ulat

ed fr

om

expe

rim

ents

car

ried

out

ove

rtw

o ye

ars

o

Cul

ture

con

diti

ons

i \

Cum

ulus

-en

clos

ed

Con

trol

Con

trol

LH

LH

LH

Den

uded

L

HL

H

* T

hese

res

ults

rep

rese

nt o

nly

the

prot

ein

synt

hesi

s.

Inhi

bito

r

Non

ea-

aman

itin

Non

ea-

aman

itin

a-am

anit

in

Non

ea-

aman

itin

inco

rpor

atio

n

Peri

od o

fin

hibi

tion

(h)

Non

e0-

18N

one

0-18

4-18

Non

e0-

18

of l

abel

led

met

hion

ine

17 9 18 9 4 7—

Num

ber

ofoo

cyte

s

B'

34 — 43 14 9 21 13

Mea

n (±

S.E

.M.)

inc

orpo

ratio

n(f

mol

oocy

te"^

"1)*

C

A

2-13

±0-

192-

75 ±

0-14

3-05

±0-

252-

62 ±

0-19

2-70

±0-

68

2-61

±0-

53—

B

'

1-99

±0-

21—

2-19

±0-

172-

18 ±

0-23

1-80

±0-

43

2-59

±0-

192-

23 ±

0-14

into

TC

A-i

nsol

uble

mat

eria

l an

d ar

e no

t in

dica

tive

of a

bsol

ute

rate

s of

O 173

t-H 00 J^

\ 50 > O 0 0 !*

Tab

le 2

. R

elat

ive

amou

nt o

f la

bell

ed p

rote

in i

n ea

ch o

f 16

mar

ker

band

s id

enti

fied

in

Fig

. 2 A

and

exp

ress

ed a

s a

perc

enta

ge o

fth

e to

tal p

rote

in s

ynth

esis

in

untr

eate

d an

d L

H-t

reat

ed e

xtra

foll

icul

ar o

ocyt

es i

n th

e pr

esen

ce a

nd a

bsen

ce o

f a-

aman

itin

. E

ach

valu

e re

pres

ents

the

mea

n of

gro

ups

(n)

of o

ocyt

es (

5 oo

cy te

st gr

oup)

inc

ubat

ed i

n [35

S] m

ethi

onin

e fo

r 3

h

Unt

reat

edU

ntre

ated

+a-

aman

itin

(0-1

8 h)

LH

-tre

ated

LH

-tre

ated

+a-

aman

itin

(0-1

8 h)

LH

-tre

ated

+a-

aman

itin

(4-1

8 h)

S.E

.M.

(poo

led)

F ra

tio*

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 n

2-38

3-10

1-95

0-44

4-22

0-60

3-49

5-50

2-65

7-21

6-94

2-94

3-0

1-94

2-80

1-76

9

1-99

2-76 96 54

3-10

4-76

1-99

6-29

7-92

3-23

2-77

2-11

2-04

1-63

3-55

3-21

0-79

1-16

2-61

1-36

3-58

4-49

2-95

7-51

6-75

1-99

2-92

2-30

3-82

2-25

1-92

2-81

2-80

0 5-23

0-16

2-94

4-20

2-12

6-61

7-41

3-31

3-20

1-91

1-99

1-42

5

* S

tati

stic

s ob

tain

ed f

rom

an

anal

ysis

of

vari

ance

for

eac

h m

arke

r ba

nd.

t In

dica

tes

band

s sh

owin

g m

arke

d he

tero

gene

ity

betw

een

grou

ps.

4-4

3-25

0-94

1-09 62 47 42 80 06 14

6-53

1-76

2-71

1-92

3-11

1-46

4

0-80

0-51

0-62

0-37

0-88

0-38

0-44

0-89

0-56

0-76

0-64

0-51

0-59

0-49

0-70

0-43

9-24

f1-

0415

-20t

13-1

6113

-961

16-8

4t2-

292-

243-

932-

483-

9010

-741

0-53

.0-8

37-

61t

3-93

a a. o o to

15

z^:

10

2A

Mr

xlO

"3

200

92 69 46

30 14-3

U)

LH

+ a

-am

aniti

n(4

-18H

)I

i:tre

ated

(0-

18 h

)

A*

-5

-4

-3

-2

LH

+ a

-am

aniti

n(0

-18

H)

LH

+

a n

ii.i

iiii

iii

|

(O-:

MD

+3

t

+5+'

6

\ \ U

ntre

ated

\ +

a-a

man

itin

^

2B

D

Fig.

2A

. F

luor

ogra

phs

of [

35S]

met

hion

ine-

labe

lled

pol

ypep

tides

fro

m (

A)

untr

eate

d oo

cyte

s, (

B)

untr

eate

d oo

cyte

s cu

lture

dfr

om e

xpla

ntat

ion

with

a-a

man

itin

, (C

) LH

-tre

ated

ooc

ytes

, (D

) LH

-tre

ated

ooc

ytes

cul

ture

d fro

m e

xpla

ntat

ion

with

a-a

man

itin

and

(E)

LH

-tre

ated

ooc

ytes

cul

ture

d w

ith a

-am

aniti

n fr

om 4

h a

fter

exp

lant

atio

n. C

umul

us-e

nclo

sed

oocy

tes

wer

e cu

lture

d fo

r18

h, l

abel

led

for

3 h

in th

e pr

esen

ce o

f 1

mC

i/m

l of

[35

S]m

ethi

onin

e, a

nd t

he la

belle

d po

lype

ptid

es s

epar

ated

by

SDS-

grad

ient

gel e

lect

roph

ores

is. 4

0000

TC

A-p

reci

pita

ble

c.p.

m. w

ere

load

ed o

nto

each

slo

t of

the

gel a

nd th

e fl

uoro

grap

hs d

evel

oped

aft

er48

h.

The

six

teen

mar

ker

band

s se

lect

ed f

or a

naly

sis

are

indi

cate

d an

d nu

mbe

red

sequ

entia

lly f

rom

the

low

to

high

rel

ativ

em

olec

ular

mas

s re

gion

s. T

he p

osit

ions

of

the 1

4 C-l

abel

led

rela

tive

mol

ecul

ar m

ass

mar

ker

prot

eins

(se

e M

ater

ials

and

Met

hods

)ar

e sh

own

on t

he r

ight

-han

d si

de.

Fig.

2B

. A

naly

sis

of t

he e

ffec

t of

a-a

man

itin

on

prot

ein

prof

iles

in u

ntre

ated

and

LH

-tre

ated

ext

rafo

llicu

lar

oocy

tes.

The

plo

tre

pres

ents

the

firs

t tw

o ca

noni

cal v

aria

tes

for

31 g

roup

s of

ooc

ytes

in th

e si

x tr

eatm

ents

. (*)

mar

ks th

e ce

ntro

id o

f ea

ch t

reat

men

tgr

oup.

O tn W o 133 z 2! D o o

RNA inhibitors and oocyte maturation 325

Table 3. Standardized 'distances', calculated as the Mahalanobis D statistic (Rao,1952), between the centroids of the treatment groups shown in Fig. 2B. The'distances' reflect the degree of difference between the patterns of protein synthesis

(see also Moor et al., 1981)

Untreated

Untreated +a-amanitin(0-18 h)

LH treated

LH treated +a-amanitin(0-18 h)

LH treated +a-amanitin(4-18 h)

Untreated

—

5-0

4-7

4-7

4-7

Untreated +a-amanitin

(0-18 h)

—

—

7-8

2-4

7-8

LH treated

—

—

—

8-0

4-6

LH treated +a-amanitin

(0-18 h)

—

—

—

—

7-6

during incubation induced numerous changes in protein synthesis in cumulus-enclosed oocytes (Fig. 2A). These differences were subjected to statisticalanalysis using the canonical variate analysis to compare the relative proportionsof labelled protein in each of 16 bands (Fig. 2A) selected previously as markersof protein change during maturation (Moor etal. 1981). From the results shownin Table 2 and from the analysis of this data (Fig. 2B), it is apparent that thepattern of protein synthesis in cumulus-enclosed oocytes cultured in the absenceof a-amanitin differs substantially from that found in oocytes cultured in thepresence of a-amanitin (see Table 3). Moreover, the analysis shows that theinhibitory effects of a-amanitin on protein synthetic changes were largely, butnot completely, overcome by delaying the addition of a-amanitin for 4h.Nevertheless, the pattern of protein synthesis still showed some differencesfrom that found in LH-treated oocytes cultured in the absence of a-amanitin,suggesting that the presence of the drug from 4-18 h of culture may have someeffect on the completion of these changes (see below).

The results of the canonical variate analyses of one-dimensional gelsdescribed above show that the changes in the patterns of polypeptide synthesiswhich occur during oocyte maturation can be suppressed by a-amanitin. Toexamine these changes in more detail and to resolve further the effects ofa-amanitin, labelled oocyte proteins were separated using two-dimensional gelelectrophoresis.

326 J. C. OSBORN AND R. M. MOOR

Analysis of acidic proteins by two-dimensional gel electrophoresisThe patterns of polypeptide synthesis of oocytes labelled from either 0 to 3 h

or from 18 to 21 h (i.e. after culture) are shown in Figs 3 and 4 respectively. Theseprofiles confirm that maturation is accompanied by major changes in the patternsof protein synthesis which involve a substantial increase of incorporation intosome polypeptides and a substantial reduction of incorporation into others.Amongst the most notable of the polypeptides which are visible before matura-tion, but which become greatly reduced during maturation are polypeptides 4(Mr 76 x 103), 13 (Mr 68 x 103), 25 (=Actin, Mr 45 x 103, band 8 on ID), 31(Mr 27-5 x 103, component of band 5 on ID), 32 (Mr 27-5 x 103, component ofband 5 on ID), 33 (Mr 25-5 x 103, band 3 on ID) and 34 (Mr 11-5 x 103). Bycontrast, although several of the major proteins synthesized by the oocyte beforematuration become prominent during maturation e.g. polypeptides 14 (Mr

16 x 103) and 29 (Mr 36-5 x 103, band 7 on ID), the majority of 'new' proteinspresent in matured oocytes were either undetectable or relatively minor polypep-tides before maturation. Proteins of this type are indicated by letters on Fig. 4 andinclude polypeptides A (Mr 135 x 103, band 15 on ID), B (Mr 67 x 103, band 10on ID), D and E (Mr 60 x 103) and L and M (Mr 28-5 x 103, band 6 on ID).

The pattern of protein synthesis of cumulus-enclosed oocytes cultured for 18 hin a-amanitin and then labelled from 18-21 h (Fig. 5) is similar to that observedin oocytes labelled from 0-3 h (Fig. 3). By contrast, oocytes cultured ina-amanitin from 4-18 h show a pattern of protein synthesis which is broadlysimilar to that observed in LH-treated oocytes cultured for 18 h in the absenceof a-amanitin (Fig. 4) but which does not show all of the changes in proteinsynthesis that accompany oocyte maturation (Fig. 6). In particular, polypeptidesC, D, E, F, G, H, L and M are either undetectable or only weakly present. Thesechanges in protein synthesis are however, observed in oocytes cultured from6-18h in a-amanitin (data not shown).

Analysis of basic proteins by two-dimensional gel electrophoresis

Although the combination of IEF in the first dimension with SDS gel

Figs 3-6. Fluorographs of two-dimensional gel separations (IEF) of[3%]methionine-labelled polypeptides from untreated oocytes labelled from 0-3 h(Fig. 3) LH-treated, cumulus-enclosed oocytes (Fig. 4), LH-treated cumulus-enclosed oocytes cultured from explantation with a-amanitin (Fig. 5) and LH-treated, cumulus-enclosed oocytes cultured with a-amanitin from 4 h after explanta-tion (Fig. 6). Oocytes were cultured for 18h (except in Fig. 3), labelled for 3h in[35S]methionine at 1 mCi/ml and the polypeptides separated by IEF followed byelectrophoresis on 15 % SDS-polyacrylamide gels. 100000 TCA-precipitable countswere applied per gel and the fluorographs developed after 3 days. In each figure,actin is indicated by the letters Ac while numbered spots enable comparisons to bemade between patterns. The spots identified by letters in Figs 4 and 6 indicate thosepolypeptides which consistently appear during maturation. The positions of the 14C-labelled relative molecular mass markers are shown on the left hand side.

RNA inhibitors and oocyte maturation 327

KS ot "

iu-4

o-4

I x-.O O—•

• 2# • 0

* * *

gfmmt^&gjuj'

co

%S2t

<2% * 7

/ 4*a* • o

1 9 . 1 * " •i

1

-• S

CM O)0) (O

(O

w O

oCOCOrr

CM0)

0)(0

CO oco co

"Figs. 3-6

328 J. C. OSBORN AND R. M. MOOR

electrophoresis in the second resolves a large number of oocyte proteins asshown in Figs 3-6, many of the basic proteins are excluded. To analyse thesynthesis of these basic proteins, we have used NEPHGE (O'Farrell et al. 1977)to resolve proteins with isoelectric points in the pH range 7-10. NEPHGEseparations of polypeptides from oocytes labelled from 0 to 3 h or from 18 to 21 hare shown in Figs 7 and 8 respectively. As in the IEF separations, the patternsshow that many polypeptides undergo major quantitative change during matura-tion. Most notable amongst these maturational changes are the reduction insynthesis of polypeptides 11 and 12 (Mr 60 x 103), 14 and 16 (Mr 51 x 103) and24 (Mr 27-5 x 103, component of band 5 on ID) and the apparent increase insynthesis of polypeptides 1 (Mr 108 x 103, band 13 on ID), 3 (Mr 96 x 103), D andE (Mr 39 x 103), K (Mr 31 x 103) and M (Mr 15 x 103, band 1 on ID). The patternof basic polypeptides synthesized by oocytes cultured continuously ina-amanitin (Fig. 9) is similar to that observed in oocytes labelled from 0 to 3h(Fig. 7). By contrast, oocytes cultured in a-amanitin from 4-18 h show an inter-mediate pattern of protein synthesis (Fig. 10). In this case, many of the polypep-tides which characterize the oocyte before maturation, such as polypeptides 11,12, 15, 16 and 17 are present at the same time as those which appear at matura-tion, e.g. polypeptides 1, 3, D, G, H, I and J. Interestingly, however, oocytescultured in a-amanitin from 6-18 h do not show this intermediate pattern (datanot shown).

a-Amanitin and denuded oocytes

Previous work has shown that the patterns of protein synthesis in denudedoocytes are qualitatively similar to those in cumulus-enclosed oocytes but thatthere are quantitative differences, the most notable being a large decrease inactin synthesis (Crosby, Osborn & Moor, 1981; Osborn & Moor, 1982). Theprofiles of labelled polypeptides illustrated in Fig. 11 confirm these results anddemonstrate that denuded oocytes cultured for 18 h in a-amanitin show a 'post-maturational pattern' of protein synthesis which is very similar to that observedin LH-treated cumulus-enclosed oocytes (Figs 2A and 4). There are, however,a number of differences between these profiles, the most notable being theabsence in denuded oocytes of polypeptides G and H, the reduction in synthesisof polypeptide 5 and the increase in synthesis of two previously minor polypep-tides (asterisks in Fig. 11D).

Site of action of a-amanitin

The results of the nuclear and protein synthesis studies suggest that the inhibit-ory action of a-amanitin on oocyte maturation is dependent upon the presenceof cumulus cells and is caused by a time-dependent inhibition of transcription.The experiments do not, however, demonstrate whether the crucial inhibitoryaction of this drug occurs within the cumulus cells or the oocyte. The ensuingstudies provide further information on the site at which a-amanitin may act.

Mr

xicr3

92-

69-

46-

30-

14-3-

RNA inhibitors and oocyte maturationIEF

329

23

27

28

22

19

"20

26 25

2625

SDS

92-

69-

46-

30-

22 2V

26

• 20

^ T

25

22

19

20, G

/ H

26 25

143-27

28 10 28

Figs 7-10. Fluorographs of two-dimensional gel separations (NEPHGE) of[3^S]methionine labelled polypeptides from untreated oocytes labelled from 0-3 h(Fig. 7), LH-treated, cumulus-enclosed oocytes (Fig. 8), LH-treated cumulus-enclosed oocytes cultured from explantation with a-amanitin (Fig. 9) and LH-treated cumulus-enclosed oocytes cultured with a-amanitin from 4 h after explanta-tion. The details of the labelling and separation of oocyte proteins are the same asin Figs 3-6 except that NEPHGE was used in the first dimension. In each figure,actin is indicated by the letters Ac while numbered spots enable comparisons to bemade between patterns. The spots identified by letters in Figs 8 and 10 indicate thosepolypeptides which appear during maturation. The positions of the 14C-labelledrelative molecular mass markers are shown on the left-hand side.

EMB73

330 J. C. OSBORN AND R. M. MOOR

Effect of a-amanitin on RNA synthesis

The uptake and incorporation of [3H]uridine into cumulus cells and oocyteshas been used as a measure of the effect of a-amanitin on RNA synthesis. Theresults from three experiments (Table 4) show firstly that both the uptake andincorporation of uridine are suppressed in a-amanitin-treated cumulus cells andthat the decrease in incorporation remains highly significant (P < 0-1) even afterthe reduced uptake is taken into consideration. Similarly, both the uptake andincorporation of uridine into oocytes were reduced by a-amanitin treatment, butin these cells, the levels were too variable to show any statistical difference fromthe controls. Nevertheless, if the apparent decline in uridine uptake into thetreated oocytes is taken into consideration and the levels of incorporation areexpressed as a ratio of the total uptake, the results show that the incorporationof [3H]uridine into a-amanitin-treated oocytes is not inhibited but may actuallybe increased.

These results suggest therefore that a-aminitin suppresses RNA synthesis inthe cumulus cells rather than in the oocytes. It should, however, be stressed that

Mrx1O"3

200-

92-

69-

46-

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14-3-

EF

id.

6 13

16 14 17 D EIt

24

2826

G H

27 \.

Ac 18 i

33 32

X1O"

92-

69-

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B C

Fig. 11. Fluorographs of [35S]methionine-labelled polypeptides from (A) LH-treated, cumulus-enclosed oocyte, (B) LH-treated denuded oocyte and (C) and (D)LH-treated, denuded oocyte cultured with a-amanitin from explantation. Oocyteswere labelled for 3h with lmCi/ml of [35S]methionine and the polypeptidesseparated by one-dimensional electrophoresis on 8-15 % linear-gradient SDS gels(A-C) and by two-dimensional electrophoresis (D) on 15 % SDS gels afterisoelectric focussing. In Fig. 11D, actin is indicated by the letters Ac while thenumbered spots enable comparisons to be made with the patterns shown in Figs 3-6.The spots identified by letters in Fig. 11D indicate those polypeptides which appearduring maturation. The two polypeptides whose synthesis is increased in denudedoocytes are marked with asterisks. The positions of the 14C-labelled relativemolecular mass markers are also shown.

SDS

RNA inhibitors and oocyte maturation 331

Table 4. The effect of a-amanitin on the uptake and incorporation of [3H]uridineinto groups of oocytes (four or five per group) and cumulus cells incubated in[3H]uridine for 4 h. Different superscripts within columns denote differences at the

0-1 % level of significance

Number of groups

Mean (± S.E.M.)

TCA-insolublec.p.m./cell

Mean (± S.E.M.)

Total c.p.m./cell

Mean (± S.E.M.)

Ratio insolublec.p.m.: Totalc.p.m. (xlO"3)

Control

14

79-3±25-6

6738±158-7

9-26±1-31

Oocytea-amanitin

14

46-6±10-1

3360±655

13-36±2-3

CumulusControl a-amanitin

19

0-43a

±0-09

3-52±0-83

161b

±18

17

0-06a

±0-02

2-23±0-6

63b

±11

with present methods, subtle changes in RNA synthesis in a-amanitin-treatedoocytes may not be detected because of the low levels of synthesis that occur evenin untreated oocytes during maturation. If this is the case, our results do notexclude the possibility that a-amanitin inhibits transcriptional activity within theoocyte.

Cumulus-cell-mediated entry of RNA inhibitors

It is known that the entry of certain substances into oocytes only occurs bydirect intercellular transmission through junctional complexes with cumuluscells (Heller & Schultz, 1980; Moor etal. 1980). The extent to which the cumuluscells facilitate the entry of one of the RNA inhibitors into the oocyte wasmeasured using radiolabelled cordycepin. The lack of radioactive a-amanitinprevented similar studies on the entry of this inhibitor. Groups of cumulus-enclosed and denuded oocytes were incubated for 3 h in 5 jUM-pHJcordycepin(specific activity 20-6Ci/mmol). After incubation, the oocytes were denuded inappropriate cases, disrupted using lO ul SDS buffer and counted using conven-tional techniques. The mean uptake of [3H]cordycepin into cumulus-enclosedand denuded oocytes was 23-5 ± 1-89 fmols per oocyte (n = 7 and8-22 ± 0-72 fmols per oocyte (n = 8) respectively. However, since an uptake of3-5 fmols per oocyte can be accounted for by the size of the extracellular space(= 0-72 nl; Moor & Smith, 1979) the corrected uptakes for cumulus-enclosed anddenuded oocytes are 20 fmols per oocyte and 4-7 fmols per oocyte respectively.

These results demonstrate that cordycepin enters the oocyte by uptake across

332 J. C. OSBORN AND R. M. MOOR

the membrane but that the rate of entry is greatly enhanced in the presence ofcumulus cells.

DISCUSSION

In this study, we have shown that the addition of a-amanitin to extrafollicularoocytes, at concentrations which suppress RNA polymerase II activity in vitro(Versteegh, Hearn & Warner, 1975) and inhibit pre-implantation embryonicdevelopment (Golbus et al. 1973; Levey et at. 1977; Braude, 1979a,b inter alia)prevents nuclear maturation and protein synthetic changes if present from theinitiation of meiosis. By contrast, this inhibitory effect is considerably reduced bydelaying the addition of the inhibitor for 1-2 h. Since a-amanitin is an effectiveinhibitor of poly A-containing RNA synthesis in mouse blastocysts (Levey &Brinster, 1978; Schindler & Sherman, 1981), our results suggest that an earlytranscriptional event is required for the resumption of meiosis in mammalianoocytes. It is, however, possible that the inhibition of maturation observed ina-amanitin-treated oocytes could have resulted from an indirect cytotoxic effectof the drug. While such secondary non-specific effects cannot be totally discoun-ted, the following observations argue against this possibility. Firstly, oocytes ex-posed to a-amanitin from 2-4 h after the initiation of meiosis complete matura-tion and undergo many of the associated changes in the pattern of proteinsynthesis. Secondly, the finding that there is no significant difference in the levelsof incorporation of [35S] methionine between untreated and a-amanitin-treatedoocytes and that only those changes in the patterns associated with maturation areaffected while other proteins appear to be resistant to a-amanitin, indicates thatprotein synthesis is not non-specifically affected by a-amanitin. Thirdly, the clearparallels between the time-dependent effects of a-amanitin and cordycepin onnuclear maturation and their reported actions on the synthesis and poly A-dependent processing of messenger RNA, make it unlikely that the two drugsshould exert the same cytotoxic effect but for differing periods of time. Wetherefore conclude that the inhibitory effects of a-amanitin and cordycepin onoocyte maturation result from a selective inhibition of transcription rather than anon-specific depression of cellular metabolism. It is uncertain, however, whetherthis transcriptional event occurs within the cumulus cells or within the oocyte.

Studies on amphibian oocytes have shown that gonadotrophin-inducedmaturation of follicle-enclosed oocytes is inhibited by actinomycin D anda-amanitin (Brachet, 1967; Wasserman & Masui, 1974) but that progesterone-induced maturation of denuded oocytes is unaffected (Baltus, Brachet, Hanocq-Quertier & Hubert, 1973; Wasserman & Masui, 1974). From these and otherresults (see Masui & Clarke, 1979) it has been concluded that, in amphibia, thegonadotrophic induction of transcriptional activity within the follicle cells affectsthe production of a progesterone-like hormone which acts on the oocyte toinduce maturation.

RNA inhibitors and oocyte maturation 333Our observations that a-amanitin inhibits [3H]uridine incorporation into

cumulus cells and that both a-amanitin and cordycepin are dependent upon thepresence of cumulus cells for their action on oocyte maturation are consistentwith the hypothesis that the inhibitors exert an indirect effect on the oocyte bysuppressing transcription within the cumulus cells. The precise mechanism bywhich transcriptional activity within the cumulus cells would affect the mam-malian oocyte is, however, unclear. It is difficult to argue convincingly that RNAsynthesized by the cumulus cells is essential for the resumption of meiosis sincethis event occurs readily in mammalian oocytes denuded of all associatedcumulus elements. Nevertheless, it is clear that uridine incorporation into thecumulus cells is significantly inhibited by a-amanitin. This suggests that eithercumulus cells synthesize relatively high amounts of mRNA and little ribosomalRNA (rRNA) or that a-amanitin indirectly suppresses the polymerase involvedin rRNA synthesis. At present, our results do not enable us to distinguish be-tween these two possibilities.

An alternative hypothesis to that outlined above postulates that a-amanitinand cordycepin inhibit transcription within the oocyte but that their passage intothe oocyte is dependent upon the presence of the cumulus cells. Our results using[3H]cordycepin support the idea that the entry of at least one of the inhibitorsinto the oocyte occurs predominantly through permeable junctions with folliclecells. There is, however, no evidence for the involvement of junction-mediatedtransmission of a-amanitin into the oocyte, although the size of the a-amanitinmolecule (919 daltons molecular mass) would not prevent its passage throughjunctions which are limited to molecules of less than 1000 daltons molecular mass(Flagg-Newton, Simpson & Loewenstein, 1979). Nevertheless, since a-amanitinhas no apparent effect on denuded oocytes (see also Crozet & Szollosi, 1980) andis known to have a low permeability into amphibian oocytes (Scheer, personalcommunication), it is likely that permeable junctions between the cumulus cellsand the oocyte also provide the means by which a-amanitin enters the oocyte.If, therefore, the importance of cumulus cells is primarily one of inhibitor trans-port, then attention should be focussed on the role of the small amount ofpoly(A)-containing RNA synthesized by fully grown oocytes (Brower et al.1981). Although our findings suggest that total RNA synthesis in oocytes is notsignificantly inhibited by a-amanitin they do not preclude the possibility that thisinhibitor selectively inhibits certain classes of RNA. The existence of ana-amanitin-sensitive RNA polymerase in oocytes of large antral follicles (Moore& Lintern-Moore, 1979) provides a means by which such an inhibition mayoccur.

The evidence obtained in the present study strongly suggests that a criticala-amanitin and cordycepin-susceptible transcriptional event within the first fewhours of maturation is a prerequisite for the sequence of nuclear and cytoplasmicchanges that occur during the resumption of meiosis. Although the intracellularlocalization of this synthetic activity has not been identified, it is clear that the

334 J. C. OSBORN AND R. M. MOOR

translation of these induced RNA species will result in the synthesis of newproteins which may be causally related to the resumption of meiosis. The ex-pectation that an a-amanitin-susceptible inductive phase of RNA synthesiswould be associated with a sensitive protein-synthetic phase is supported by theobservations that protein synthesis is only required for the first 2h after LH-induced meiosis in intact rat follicles in vitro (Lindner et al. 1974) or for the first9h in extrafollicular sheep oocytes (Moor & Polge, unpublished observations).However, further interpretation of this data is complicated by the belief that,since the treatment of extrafollicular mouse oocytes with puromycin andcycloheximide arrests meiosis at the prometaphase I stage but fails to inhibitGVBD (Stern, Rayvis & Kennedy, 1972; Golbus & Stein, 1976; Wassarman &Letourneau, 19766; Schultz & Wassarman, 1911b), concomitant proteinsynthesis is not required for the resumption of meiosis. By contrast, recentexperiments have shown that the pretreatment of follicle-enclosed oocytes withpuromycin before isolation and culture with puromycin, significantly reducedthe rate of GVBD from 95% to 35% (Ekholm & Magnusson, 1979). Oneexplanation for these results is that protein synthesis is necessary for GVBD, butthat problems associated with the penetration of puromycin could account for itsinability to block meiosis in earlier reports. Nevertheless, Ekholm & Magnusson(1979) conclude that their results indicate the existence of short-lived proteinsnecessary for the resumption of meiosis. Pretreatment with puromycin wouldthen lead to the depletion of these proteins and in the continuous presence ofpuromycin, GVBD would not occur. Such hypothetical short-lived proteins maybe analagous to the tyrosine-rich short-lived proteins shown by Mangia &Canipari (1977) to be synthesized during the first 3 h of meiosis in the mouseoocyte and thought to be involved in the regulation of early meiotic events. Thedetection of changes in the pattern of protein synthesis prior to GVBD(McGaughey & Van Blerkom, 1977; Schultz & Wassarman, 1977a; Van Bler-kom & McGaughey, 1978; Wassarman, Schultz & Letourneau, 1979) and theaccumulation of newly synthesized proteins in the germinal vesicle (Wassarman& Letourneau, 1916b; Motlik, Kopecny & Pivko, 1978) suggests that such earlyproteins could indeed have specific functions in the control of meiotic matura-tion. However, further research is necessary both to provide definitive evidenceon the role of short-lived proteins in the control of meiosis and to determinewhether these protein changes are transcriptionally dependent.

We have shown previously that significant qualitative and quantitativechanges in protein synthesis occur in intrafollicular oocytes matured in vitro(Moor et al. 1981) and that similar changes occur in extrafollicular oocytes(Crosby, Osborn & Moor, 1981). In the present paper we have used two-dimensional gel electrophoresis (IEF and NEPHGE) to analyse these changesin more detail. Our results confirm that major changes in the synthesis of bothacidic and basic proteins occur during oocyte maturation and that the presenceof a-amanitin for the first 4 h after the induction of meiosis effectively blocks

RNA inhibitors and oocyte maturation 335these changes. However, it is clear from both the canonical variate and two-dimensional gel analyses that the completion of the changes in protein synthesisis affected by the continued presence of a-amanitin from 4-18 h of culture eventhough the resumption of meiosis is not inhibited. The finding that such inter-mediate patterns of polypeptide synthesis do not occur when the addition ofa-amanitin is delayed for 6 h suggests that changes in the synthesis of certainpolypeptides during maturation are dependent upon a longer period of transcrip-tional activity but that the resumption of meiosis is dependent upon RNAssynthesized at the beginning of maturation.

Finally, it is well documented that the process of meiotic maturation is ac-companied by marked changes in the patterns of protein synthesis (Schultz &Wassarman, 1977a,b; Schultz, Letourneau & Wassarman, 1978; Warnes, Moor& Johnson, 1977; Van Blerkom & McGaughey, 1978) and it has been claimedthat this reprogramming of protein synthesis is dependent upon the mixing of theoocytes 'nucleoplasm' and cytoplasm resulting in the mobilization of preformedmRNAs stored in the cytoplasm (Schultz & Wassarman, I977a,b; Schultz et al.1978). While a causal relationship between these two events has still to be deter-mined, our experiments show that both GVBD and the changes in proteinsynthesis which characterize maturation in sheep oocytes are dependent upon aninitial transcriptional event at the onset of maturation which precedes GVBD.We are therefore unable to support the view that RNA synthesis is not necessaryfor the resumption of meiosis in mammalian oocytes.

We thank Mr Ian Crosby for his skilled technical assistance and Dr D. E. Walters of theA.R.C. Statistical Group, Department of Applied Biology, Cambridge for his advice on thestatistical analysis. The purified gonadotrophin used in this study was generously donated bythe National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes ofHealth, Bethesda, Maryland. One of us (J. C. Osborn) is indebted to the Medical ResearchCouncil for financial support.

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{Accepted 26 July 1982)