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J. Embryol. exp. Morph. 73, 317-338, 1983
Printed in Great Britain © The Company of Biologists Limited 1983
Time-dependent effects of a-amanitin on nuclear
maturation and protein synthesis in mammalian
oocytes
By J. C. OSBORN 1 AND R. M. MOOR 1
From the A.R. C. Institute of Animal Physiology, Cambridge
SUMMARY
The addition of a-amanitin to extrafollicular, cumulus-enclosed ovine oocytes at explantation inhibits meiotic maturation and prevents many of the changes in protein synthesis that
normally accompany maturation. By contrast, these inhibitory effects are considerably
reduced by either delaying the addition of the drug for 1-4 h or by denuding the oocytes of all
associated cumulus cells at the onset of culture. The observations that the inhibitory effect of
cordycepin on nuclear maturation is also time-dependent and cumulus-cell-dependent and
that the oocyte is susceptible to cordycepin for longer than its sensitivity to a-amanitin are
consistent 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 the
initiation of those changes in protein synthesis required for the regulation of nuclear and
cytoplasmic maturation. It is uncertain, however, whether this transcriptional event occurs
within the cumulus cells or within the oocyte.
INTRODUCTION
Although most of the RNA present in mammalian oocytes is synthesized and
accumulated 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 to
within 1 h of germinal vesicle breakdown (GVBD) and that some of the newly
synthesized RNA is released into the cytoplasm before GVBD (Bloom & Mukherjee, 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 completion of the first meiotic division. However, other results show that meiosis is
not 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, Cambridge
CB3 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) at
low concentrations (Manes, 1973) and at higher concentrations has deleterious
side effects on protein synthesis, respiration and glycolysis (Honig & Rabinovitz,
1965; Laszlo, Miller, McCarthy & Hochstein, 1966), the suppression of meiotic
maturation by actinomycin D has been regarded with some caution. A more
specific 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 particular
drug has been shown to be an efficient inhibitor of development in preimplantation rabbit (Van Blerkom, 1977) and mouse embryos (Golbus, Calarco & Epstein, 1973; Warner & Versteegh, 1974; Levey, Troike & Brinster, 1977;
Braude, 1979a,6) at concentrations which completely inhibit RNA polymerase
II activity in vitro (Versteegh, Hearn & Warner, 1975). We have therefore made
use of this selective action of a-amanitin to determine whether new mRNA
synthesis is required for the initiation of either nuclear or cytoplasmic events
during 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 cycle
with 1250 i.u. of pregnant mare serum gonadotrophin and slaughtered 40 h later.
Intact, non-atretic follicles were dissected from the ovaries at room temperature
and opened to remove the entire cumulus-oocyte complex. Two types of culture
were carried out: (i) the intact cumulus-oocyte complex was cultured (cumulusenclosed oocytes) or (ii) the oocyte was cultured after removal of the cumulus
cells with fine pipettes (denuded oocytes). Cumulus-enclosed and denuded
oocytes were cultured at 37 °C in media containing 10 /ig ml" 1 NIH-LH-S18 using
the conditions and culture medium described by Crosby, Osborn & Moor (1981).
For cultures with a-amanitin and cordycepin, cumulus-enclosed and denuded
oocytes were divided into groups and exposed to a-amanitin (BoeringherMannheim, 10/igml"1) or cordycepin (Sigma, SOjUgml"1) at selected times after
removal from the follicle (Figs 1A and B). In those groups of oocytes cultured
with a-amanitin or cordycepin from explantation (oh) all preparative procedures
were carried out in media containing the appropriate inhibitor. After 18 to 24 h
culture, cumulus-enclosed and denuded oocytes were either examined as whole
mounts after staining with lacmoid or radiolabelled with [35S]methionine for
one- and two-dimensional gel electrophoresis.
Radiolabelling of oocytes
Groups of six to ten denuded or cumulus-enclosed oocytes were labelled at
37 °C for 3h in 50 \x\ of incubation medium (Moor, Smith & Dawson, 1980)
RNA inhibitors and oocyte maturation
319
35
containing either 500/iCi or lmCi/ml [ S]methionine (Specific activity >
lOOOCi/mmol, Radiochemical Centre, Amersham). After incubation, groups
of denuded and cumulus-enclosed oocytes were washed once in incubation
medium and the latter were denuded of cumulus cells. Denuded oocytes were
then briefly washed in lOmM-Tris-HCl, pH7-4, collected in a small volume of
Tris buffer (<5/A), lyophylized and frozen at - 7 0 °C until required for
electrophoresis.
Electrophoretic analysis of oocyte proteins
Labelled oocyte polypeptides were analysed in one dimension as described by
Moor, Osborn, Cran & Walters (1981), or in two dimensions essentially according to O'Farrell (1975) and O'Farrell, Goodman & O'Farrell (1977).
For one-dimensional analysis, groups of oocytes were lyzed in 25-30/il of
sample buffer (O'Farrell, 1975) and a 5/il aliquot used for determining incorporation of radioactivity into TCA-precipitable material. Equal numbers of
TCA-precipitable counts were applied to a 8-15 % linear gradient SDS
polyacrylamide slab gel and the polypeptides separated for 3h at a constant
current of 20 mA per gel. Labelled proteins were visualized by fluorography
(Bonner & Laskey, 1974) using preflashed' Kodak X-Omat film at - 7 0 °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. Microdensitometer scans were made of each fluorogram and a quantitative and statistical
analysis of the changes in protein synthesis carried out as described by Moor et
al. (1981).
For two-dimensional (2D) analysis of acidic and basic polypeptides, groups of
oocytes were placed in 20/^1 of lysis buffer containing 9-5M-urea, 2% w/v
Nonidet P40 (Sigma), 5 % mercaptoethanol and 2% ampholines (1-6% pH
range 5-7 and 0-4% pH range 3-5-10; LKB). After freezing and thawing the
samples twice, duplicate 1 jwl aliquots were used to determine the TCAprecipitable counts. Samples containing 100 000 c.p.m. in 15 /il were applied to
4 % 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 isoelectric
focussing (IEF) and 2 % ampholines (1 % pH range 7-9 and 1 % pH range 8-9-5)
for non-equilibrium pH gradient electrophoresis (NEPHGE). After electrophoresis at 400V for 18 h (IEF) or 4|h at 400V (NEPHGE) the gels were
equilibrated for 20 min in sample buffer before loading onto 15 % SDS
polyacrylamide slab gels. After electrophoresis, the gels were processed for
fluorography and exposed to preflashed Kodak X-Omat H film for 3 days. The
molecular weights of unknown proteins in the second dimension gels were determined by comparison with concurrently electrophoresed 14C-marker proteins
(see above) added to the agarose bed upon which the IEF or NEPHGE gel was
placed.
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 incubation
medium 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 Tris
buffer and disrupted in 30 /il SDS sample buffer. Duplicate 2-5 /A aliquots of each
sample were used to determine total counts and the remainder of each sample
used 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 288
oocytes cultured in a-amanitin at various times after explantation. Fig. 1A shows
that 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 formation
of a metaphase plate had occurred. By contrast, the inhibitory effect of
a-amanitin on meiotic maturation was greatly decreased by delaying the addition
of the inhibitor to cumulus-enclosed oocytes or by culturing the oocytes in the
absence of cumulus cells. Thus, when a-amanitin was added at either 1 h or 2h
after explantation, 60 % and 83 % respectively of cumulus-enclosed oocytes
underwent GVBD, while 73 % of denuded oocytes, cultured from explantation
with a-amanitin, showed normal metaphase plates. These results demonstrate
that the maintenance of oocyte-cumulus cell contact is necessary for the inhibitory action of a-amanitin on nuclear maturation, but that the cumulus-enclosed
oocyte is only susceptible to the inhibitor for a short period after the initiation
of meiosis.
To confirm the specificity of action of a-amanitin, we have used a second
inhibitor, cordycepin, which blocks the post-transcriptional adenylation of
nuclear RNAs (Darnell, Philipson, Wall & Adesnik, 1971; Penman, Rosbash &
Penman, 1970). Fig. IB shows that the effects of cordycepin on nuclear maturation are very similar to those obtained with a-amanitin being both time dependent and cumulus-cell dependent. In addition, the finding that the cumulusenclosed oocyte is susceptible to cordycepin for longer than its sensitivity to
a-amanitin is consistent with the differential effects of these drugs on the transcription and processing of RNA.
a-amanitin and protein synthesis
The results presented in Table 1 show that the incorporation of labelled
methionine into TCA-insoluble material in cumulus-enclosed and denuded
oocytes is unaffected by a-amanitin. By contrast, the presence of a-amanitin
RNA inhibitors and oocyte maturation
321
GV
100
(20)
Metaphase
(96)
80
(30)
(45)
(55)
(42)
60
40
20
Untreated
Oh
lh
m
2h
Denuded, Oh
6h
Onset of a-amanitin treatment
%
100
GV
(38)
(15)
Metaphase
(40)
80
(15)
(11)
(12)
60
40
20
Untreated
Oh
4h
5h
6h
Denuded, Oh
Onset of cordycepin treatment
Fig. 1. Nuclear development of cumulus-enclosed denuded oocytes examined 18 h
after (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 metaphase
stages of development. Figures in parentheses indicate number of oocytes examined.
LH
LH
LH
LH
LH
Control
Control
\
None
a-amanitin
None
a-amanitin
a-amanitin
None
a-amanitin
Inhibitor
None
0-18
None
0-18
4-18
None
0-18
(h)
Period of
inhibition
7
—
17
9
18
9
4
21
13
34
—
43
14
9
B'
A
—
2-13 ±0-19
2-75 ±0-14
3-05 ±0-25
2-62 ±0-19
2-70 ±0-68
2-61 ±0-53
C
0
0
!*
O
—
>
50
J^\
173
t-H
00
O
o
2-19 ±0-17
2-18 ±0-23
1-80 ±0-43
2-59 ±0-19
2-23 ±0-14
1-99 ±0-21
B '
Mean (± S.E.M.) incorporation
(fmoloocyte"^" 1 )*
* These results represent only the incorporation of labelled methionine into TCA-insoluble material and are not indicative of absolute rates of
protein synthesis.
Denuded
Cumulusenclosed
i
Culture conditions
Number of
oocytes
Table 1. Incorporation of [35S] methionine into cumulus-enclosed and denuded oocytes after 18 h culture in the presence and
absence of a-amanitin (10 p.gml~l). A and B represent the levels of incorporation calculated from experiments carried out over
two years
OJ
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
3-10
4-76
1-99
6-29
7-92
3-23
2-77
2-11
2-04
1-63
54
1-99
2-76
96
Untreated +
a-amanitin
(0-18 h)
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
LH-treated
LH-treated +
a-amanitin
(4-18 h)
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
LH-treated +
a-amanitin
(0-18 h)
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
* Statistics obtained from an analysis of variance for each marker band.
t Indicates bands showing marked heterogeneity between groups.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
n
Untreated
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
(pooled)
S.E.M.
9-24f
1-04
15-20t
13-161
13-961
16-84t
2-29
2-24
3-93
2-48
3-90
10-741
0-53
.0-83
7-61t
3-93
F ratio*
Table 2. Relative amount of labelled protein in each of 16 marker bands identified in Fig. 2 A and expressed as a percentage of
the total protein synthesis in untreated and LH-treated extrafollicular oocytes in the presence and absence of a-amanitin. Each
value represents the mean of groups (n) of oocytes (5 oocy testgroup) incubated in [35S] methionine for 3 h
to
o
o
a
a.
10
2A
15 z^:
2B
-5
-4
-3
nii.iiiiiiii
A*
(O-:MD
LH + a
-2
LH + a-amanitin
(4-18H)
|
I i:treated (0-18 h)
t
+3
\
+'6
^
\ Untreated
\ + a-amanitin
+5
LH + a-amanitin
(0-18 H)
Fig. 2A. Fluorographs of [35S] methionine-labelled polypeptides from (A) untreated oocytes, (B) untreated oocytes cultured
from explantation with a-amanitin, (C) LH-treated oocytes, (D) LH-treated oocytes cultured from explantation with a-amanitin
and (E) LH-treated oocytes cultured with a-amanitin from 4 h after explantation. Cumulus-enclosed oocytes were cultured for
18 h, labelled for 3 h in the presence of 1 mCi/ml of [35S]methionine, and the labelled polypeptides separated by SDS-gradient
gel electrophoresis. 40000 TCA-precipitable c.p.m. were loaded onto each slot of the gel and the fluorographs developed after
48 h. The sixteen marker bands selected for analysis are indicated and numbered sequentially from the low to high relative
molecular mass regions. The positions of the 14C-labelled relative molecular mass marker proteins (see Materials and Methods)
are shown on the right-hand side.
Fig. 2B. Analysis of the effect of a-amanitin on protein profiles in untreated and LH-treated extrafollicular oocytes. The plot
represents the first two canonical variates for 31 groups of oocytes in the six treatments. (*) marks the centroid of each treatment
group.
D
14-3
30
46
69
92
200
Mr
xlO"3
o
o
2!
D
z
133
o
O
tn
W
U)
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)
Untreated
—
—
—
—
Untreated +
a-amanitin
(0-18 h)
5-0
—
—
—
LH treated
4-7
7-8
—
—
LH treated +
a-amanitin
(0-18 h)
4-7
2-4
8-0
—
LH treated +
a-amanitin
(4-18 h)
4-7
7-8
4-6
7-6
during incubation induced numerous changes in protein synthesis in cumulusenclosed oocytes (Fig. 2A). These differences were subjected to statistical
analysis using the canonical variate analysis to compare the relative proportions
of labelled protein in each of 16 bands (Fig. 2A) selected previously as markers
of protein change during maturation (Moor etal. 1981). From the results shown
in Table 2 and from the analysis of this data (Fig. 2B), it is apparent that the
pattern of protein synthesis in cumulus-enclosed oocytes cultured in the absence
of a-amanitin differs substantially from that found in oocytes cultured in the
presence of a-amanitin (see Table 3). Moreover, the analysis shows that the
inhibitory effects of a-amanitin on protein synthetic changes were largely, but
not completely, overcome by delaying the addition of a-amanitin for 4h.
Nevertheless, the pattern of protein synthesis still showed some differences
from 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 some
effect on the completion of these changes (see below).
The results of the canonical variate analyses of one-dimensional gels
described above show that the changes in the patterns of polypeptide synthesis
which occur during oocyte maturation can be suppressed by a-amanitin. To
examine these changes in more detail and to resolve further the effects of
a-amanitin, labelled oocyte proteins were separated using two-dimensional gel
electrophoresis.
326
J. C. OSBORN AND R. M. MOOR
Analysis of acidic proteins by two-dimensional gel electrophoresis
The 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. These
profiles confirm that maturation is accompanied by major changes in the patterns
of protein synthesis which involve a substantial increase of incorporation into
some polypeptides and a substantial reduction of incorporation into others.
Amongst the most notable of the polypeptides which are visible before maturation, 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 of
band 5 on ID), 33 (Mr 25-5 x 103, band 3 on ID) and 34 (Mr 11-5 x 103). By
contrast, although several of the major proteins synthesized by the oocyte before
maturation 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' proteins
present in matured oocytes were either undetectable or relatively minor polypeptides before maturation. Proteins of this type are indicated by letters on Fig. 4 and
include polypeptides A (Mr 135 x 103, band 15 on ID), B (Mr 67 x 103, band 10
on 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 h
in a-amanitin and then labelled from 18-21 h (Fig. 5) is similar to that observed
in oocytes labelled from 0-3 h (Fig. 3). By contrast, oocytes cultured in
a-amanitin from 4-18 h show a pattern of protein synthesis which is broadly
similar to that observed in LH-treated oocytes cultured for 18 h in the absence
of a-amanitin (Fig. 4) but which does not show all of the changes in protein
synthesis that accompany oocyte maturation (Fig. 6). In particular, polypeptides
C, D, E, F, G, H, L and M are either undetectable or only weakly present. These
changes in protein synthesis are however, observed in oocytes cultured from
6-18h 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 cumulusenclosed oocytes cultured from explantation with a-amanitin (Fig. 5) and LHtreated, cumulus-enclosed oocytes cultured with a-amanitin from 4 h after explantation (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 by
electrophoresis on 15 % SDS-polyacrylamide gels. 100000 TCA-precipitable counts
were 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 be
made between patterns. The spots identified by letters in Figs 4 and 6 indicate those
polypeptides which consistently appear during maturation. The positions of the 14Clabelled relative molecular mass markers are shown on the left hand side.
RNA inhibitors and oocyte maturation
327
S o
K
t "
iu-4
o-4
*
I
O
* *
x-.
O—•
•2
#•0
gfmmt^&gjuj'
co
t
%S2
9.1* " •
1
<2% * 7
i
/ 4*
a* •
1
o
-•
CM
0)
w
O
O)
(O
(O
o
CO
CO
CM
rr
0)
"Figs. 3-6
0)
(0
S
CO
o
co
co
328
J. C. OSBORN AND R. M. MOOR
electrophoresis in the second resolves a large number of oocyte proteins as
shown in Figs 3-6, many of the basic proteins are excluded. To analyse the
synthesis 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. NEPHGE
separations of polypeptides from oocytes labelled from 0 to 3 h or from 18 to 21 h
are shown in Figs 7 and 8 respectively. As in the IEF separations, the patterns
show that many polypeptides undergo major quantitative change during maturation. Most notable amongst these maturational changes are the reduction in
synthesis of polypeptides 11 and 12 (Mr 60 x 103), 14 and 16 (Mr 51 x 103) and
24 (Mr 27-5 x 103, component of band 5 on ID) and the apparent increase in
synthesis of polypeptides 1 (Mr 108 x 103, band 13 on ID), 3 (Mr 96 x 103), D and
E (Mr 39 x 103), K (Mr 31 x 103) and M (Mr 15 x 103, band 1 on ID). The pattern
of basic polypeptides synthesized by oocytes cultured continuously in
a-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 intermediate pattern of protein synthesis (Fig. 10). In this case, many of the polypeptides 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 maturation, e.g. polypeptides 1, 3, D, G, H, I and J. Interestingly, however, oocytes
cultured in a-amanitin from 6-18 h do not show this intermediate pattern (data
not shown).
a-Amanitin and denuded oocytes
Previous work has shown that the patterns of protein synthesis in denuded
oocytes are qualitatively similar to those in cumulus-enclosed oocytes but that
there are quantitative differences, the most notable being a large decrease in
actin synthesis (Crosby, Osborn & Moor, 1981; Osborn & Moor, 1982). The
profiles of labelled polypeptides illustrated in Fig. 11 confirm these results and
demonstrate that denuded oocytes cultured for 18 h in a-amanitin show a 'postmaturational pattern' of protein synthesis which is very similar to that observed
in LH-treated cumulus-enclosed oocytes (Figs 2A and 4). There are, however,
a number of differences between these profiles, the most notable being the
absence in denuded oocytes of polypeptides G and H, the reduction in synthesis
of polypeptide 5 and the increase in synthesis of two previously minor polypeptides (asterisks in Fig. 11D).
Site of action of a-amanitin
The results of the nuclear and protein synthesis studies suggest that the inhibitory action of a-amanitin on oocyte maturation is dependent upon the presence
of cumulus cells and is caused by a time-dependent inhibition of transcription.
The experiments do not, however, demonstrate whether the crucial inhibitory
action of this drug occurs within the cumulus cells or the oocyte. The ensuing
studies provide further information on the site at which a-amanitin may act.
RNA inhibitors and oocyte maturation
Mr
xicr3
329
IEF
926946-
19
"20
22
23
30-
25
26
25
14-3-
26
27
28
SDS
92-
^T
69-
19
4622
2V
• 20
20
,G
/H
22
3025
26
26
25
14327
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 cumulusenclosed oocytes cultured from explantation with a-amanitin (Fig. 9) and LHtreated cumulus-enclosed oocytes cultured with a-amanitin from 4 h after explantation. The details of the labelling and separation of oocyte proteins are the same as
in 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 be
made between patterns. The spots identified by letters in Figs 8 and 10 indicate those
polypeptides which appear during maturation. The positions of the 14C-labelled
relative 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 oocytes
has been used as a measure of the effect of a-amanitin on RNA synthesis. The
results from three experiments (Table 4) show firstly that both the uptake and
incorporation of uridine are suppressed in a-amanitin-treated cumulus cells and
that the decrease in incorporation remains highly significant (P < 0-1) even after
the reduced uptake is taken into consideration. Similarly, both the uptake and
incorporation of uridine into oocytes were reduced by a-amanitin treatment, but
in these cells, the levels were too variable to show any statistical difference from
the controls. Nevertheless, if the apparent decline in uridine uptake into the
treated oocytes is taken into consideration and the levels of incorporation are
expressed as a ratio of the total uptake, the results show that the incorporation
of [3H]uridine into a-amanitin-treated oocytes is not inhibited but may actually
be increased.
These results suggest therefore that a-aminitin suppresses RNA synthesis in
the cumulus cells rather than in the oocytes. It should, however, be stressed that
Mr
x1O" 3
EF
X1O"
92-
200-
6
69-
13
16 14
D
17
24
69-
46-
Ac
GH
28
26
27
E
It
92-
18
i
\.
SDS
463033
30-
32
id.
14-314-3B
C
Fig. 11. Fluorographs of [35S]methionine-labelled polypeptides from (A) LHtreated, cumulus-enclosed oocyte, (B) LH-treated denuded oocyte and (C) and (D)
LH-treated, denuded oocyte cultured with a-amanitin from explantation. Oocytes
were labelled for 3h with lmCi/ml of [35S]methionine and the polypeptides
separated by one-dimensional electrophoresis on 8-15 % linear-gradient SDS gels
(A-C) and by two-dimensional electrophoresis (D) on 15 % SDS gels after
isoelectric focussing. In Fig. 11D, actin is indicated by the letters Ac while the
numbered 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 appear
during maturation. The two polypeptides whose synthesis is increased in denuded
oocytes are marked with asterisks. The positions of the 14C-labelled relative
molecular mass markers are also shown.
RNA inhibitors and oocyte maturation
331
Table 4. The effect of a-amanitin on the uptake and incorporation of [3H]uridine
into 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
Control
Number of groups
14
Mean (± S.E.M.)
TCA-insoluble
c.p.m./cell
79-3
±25-6
Mean (± S.E.M.)
Total c.p.m./cell
6738
±158-7
Mean (± S.E.M.)
Ratio insoluble
c.p.m.: Total
c.p.m. (xlO" 3 )
9-26
±1-31
Oocyte
a-amanitin
14
46-6
±10-1
3360
±655
13-36
±2-3
Cumulus
a-amanitin
Control
19
17
a
0-43
±0-09
0-06a
±0-02
3-52
±0-83
2-23
±0-6
161b
±18
63b
±11
with present methods, subtle changes in RNA synthesis in a-amanitin-treated
oocytes may not be detected because of the low levels of synthesis that occur even
in untreated oocytes during maturation. If this is the case, our results do not
exclude the possibility that a-amanitin inhibits transcriptional activity within the
oocyte.
Cumulus-cell-mediated entry of RNA inhibitors
It is known that the entry of certain substances into oocytes only occurs by
direct intercellular transmission through junctional complexes with cumulus
cells (Heller & Schultz, 1980; Moor etal. 1980). The extent to which the cumulus
cells facilitate the entry of one of the RNA inhibitors into the oocyte was
measured using radiolabelled cordycepin. The lack of radioactive a-amanitin
prevented similar studies on the entry of this inhibitor. Groups of cumulusenclosed and denuded oocytes were incubated for 3 h in 5 jUM-pHJcordycepin
(specific activity 20-6Ci/mmol). After incubation, the oocytes were denuded in
appropriate cases, disrupted using lO^ul SDS buffer and counted using conventional techniques. The mean uptake of [3H]cordycepin into cumulus-enclosed
and denuded oocytes was 23-5 ± 1-89 fmols per oocyte (n = 7 and
8-22 ± 0-72 fmols per oocyte (n = 8) respectively. However, since an uptake of
3-5 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 and
denuded 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 of
cumulus cells.
DISCUSSION
In this study, we have shown that the addition of a-amanitin to extrafollicular
oocytes, at concentrations which suppress RNA polymerase II activity in vitro
(Versteegh, Hearn & Warner, 1975) and inhibit pre-implantation embryonic
development (Golbus et al. 1973; Levey et at. 1977; Braude, 1979a,b inter alia)
prevents nuclear maturation and protein synthetic changes if present from the
initiation of meiosis. By contrast, this inhibitory effect is considerably reduced by
delaying the addition of the inhibitor for 1-2 h. Since a-amanitin is an effective
inhibitor of poly A-containing RNA synthesis in mouse blastocysts (Levey &
Brinster, 1978; Schindler & Sherman, 1981), our results suggest that an early
transcriptional event is required for the resumption of meiosis in mammalian
oocytes. It is, however, possible that the inhibition of maturation observed in
a-amanitin-treated oocytes could have resulted from an indirect cytotoxic effect
of the drug. While such secondary non-specific effects cannot be totally discounted, the following observations argue against this possibility. Firstly, oocytes exposed to a-amanitin from 2-4 h after the initiation of meiosis complete maturation and undergo many of the associated changes in the pattern of protein
synthesis. Secondly, the finding that there is no significant difference in the levels
of incorporation of [35S] methionine between untreated and a-amanitin-treated
oocytes and that only those changes in the patterns associated with maturation are
affected while other proteins appear to be resistant to a-amanitin, indicates that
protein synthesis is not non-specifically affected by a-amanitin. Thirdly, the clear
parallels between the time-dependent effects of a-amanitin and cordycepin on
nuclear maturation and their reported actions on the synthesis and poly Adependent processing of messenger RNA, make it unlikely that the two drugs
should exert the same cytotoxic effect but for differing periods of time. We
therefore conclude that the inhibitory effects of a-amanitin and cordycepin on
oocyte maturation result from a selective inhibition of transcription rather than a
non-specific depression of cellular metabolism. It is uncertain, however, whether
this transcriptional event occurs within the cumulus cells or within the oocyte.
Studies on amphibian oocytes have shown that gonadotrophin-induced
maturation of follicle-enclosed oocytes is inhibited by actinomycin D and
a-amanitin (Brachet, 1967; Wasserman & Masui, 1974) but that progesteroneinduced maturation of denuded oocytes is unaffected (Baltus, Brachet, HanocqQuertier & Hubert, 1973; Wasserman & Masui, 1974). From these and other
results (see Masui & Clarke, 1979) it has been concluded that, in amphibia, the
gonadotrophic induction of transcriptional activity within the follicle cells affects
the production of a progesterone-like hormone which acts on the oocyte to
induce maturation.
RNA inhibitors and oocyte maturation
333
3
Our observations that a-amanitin inhibits [ H]uridine incorporation into
cumulus cells and that both a-amanitin and cordycepin are dependent upon the
presence of cumulus cells for their action on oocyte maturation are consistent
with the hypothesis that the inhibitors exert an indirect effect on the oocyte by
suppressing transcription within the cumulus cells. The precise mechanism by
which transcriptional activity within the cumulus cells would affect the mammalian oocyte is, however, unclear. It is difficult to argue convincingly that RNA
synthesized by the cumulus cells is essential for the resumption of meiosis since
this event occurs readily in mammalian oocytes denuded of all associated
cumulus elements. Nevertheless, it is clear that uridine incorporation into the
cumulus cells is significantly inhibited by a-amanitin. This suggests that either
cumulus cells synthesize relatively high amounts of mRNA and little ribosomal
RNA (rRNA) or that a-amanitin indirectly suppresses the polymerase involved
in rRNA synthesis. At present, our results do not enable us to distinguish between these two possibilities.
An alternative hypothesis to that outlined above postulates that a-amanitin
and cordycepin inhibit transcription within the oocyte but that their passage into
the 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 inhibitors
into the oocyte occurs predominantly through permeable junctions with follicle
cells. There is, however, no evidence for the involvement of junction-mediated
transmission of a-amanitin into the oocyte, although the size of the a-amanitin
molecule (919 daltons molecular mass) would not prevent its passage through
junctions which are limited to molecules of less than 1000 daltons molecular mass
(Flagg-Newton, Simpson & Loewenstein, 1979). Nevertheless, since a-amanitin
has no apparent effect on denuded oocytes (see also Crozet & Szollosi, 1980) and
is known to have a low permeability into amphibian oocytes (Scheer, personal
communication), it is likely that permeable junctions between the cumulus cells
and 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 transport, then attention should be focussed on the role of the small amount of
poly(A)-containing RNA synthesized by fully grown oocytes (Brower et al.
1981). Although our findings suggest that total RNA synthesis in oocytes is not
significantly inhibited by a-amanitin they do not preclude the possibility that this
inhibitor selectively inhibits certain classes of RNA. The existence of an
a-amanitin-sensitive RNA polymerase in oocytes of large antral follicles (Moore
& Lintern-Moore, 1979) provides a means by which such an inhibition may
occur.
The evidence obtained in the present study strongly suggests that a critical
a-amanitin and cordycepin-susceptible transcriptional event within the first few
hours of maturation is a prerequisite for the sequence of nuclear and cytoplasmic
changes that occur during the resumption of meiosis. Although the intracellular
localization 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 new
proteins which may be causally related to the resumption of meiosis. The expectation that an a-amanitin-susceptible inductive phase of RNA synthesis
would be associated with a sensitive protein-synthetic phase is supported by the
observations that protein synthesis is only required for the first 2h after LHinduced meiosis in intact rat follicles in vitro (Lindner et al. 1974) or for the first
9h 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 and
cycloheximide arrests meiosis at the prometaphase I stage but fails to inhibit
GVBD (Stern, Rayvis & Kennedy, 1972; Golbus & Stein, 1976; Wassarman &
Letourneau, 19766; Schultz & Wassarman, 1911b), concomitant protein
synthesis is not required for the resumption of meiosis. By contrast, recent
experiments have shown that the pretreatment of follicle-enclosed oocytes with
puromycin before isolation and culture with puromycin, significantly reduced
the rate of GVBD from 95% to 3 5 % (Ekholm & Magnusson, 1979). One
explanation for these results is that protein synthesis is necessary for GVBD, but
that problems associated with the penetration of puromycin could account for its
inability to block meiosis in earlier reports. Nevertheless, Ekholm & Magnusson
(1979) conclude that their results indicate the existence of short-lived proteins
necessary for the resumption of meiosis. Pretreatment with puromycin would
then lead to the depletion of these proteins and in the continuous presence of
puromycin, GVBD would not occur. Such hypothetical short-lived proteins may
be 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 mouse
oocyte and thought to be involved in the regulation of early meiotic events. The
detection of changes in the pattern of protein synthesis prior to GVBD
(McGaughey & Van Blerkom, 1977; Schultz & Wassarman, 1977a; Van Blerkom & McGaughey, 1978; Wassarman, Schultz & Letourneau, 1979) and the
accumulation of newly synthesized proteins in the germinal vesicle (Wassarman
& Letourneau, 1916b; Motlik, Kopecny & Pivko, 1978) suggests that such early
proteins could indeed have specific functions in the control of meiotic maturation. However, further research is necessary both to provide definitive evidence
on the role of short-lived proteins in the control of meiosis and to determine
whether these protein changes are transcriptionally dependent.
We have shown previously that significant qualitative and quantitative
changes 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 twodimensional gel electrophoresis (IEF and NEPHGE) to analyse these changes
in more detail. Our results confirm that major changes in the synthesis of both
acidic and basic proteins occur during oocyte maturation and that the presence
of a-amanitin for the first 4 h after the induction of meiosis effectively blocks
RNA inhibitors and oocyte maturation
335
these changes. However, it is clear from both the canonical variate and twodimensional gel analyses that the completion of the changes in protein synthesis
is affected by the continued presence of a-amanitin from 4-18 h of culture even
though the resumption of meiosis is not inhibited. The finding that such intermediate patterns of polypeptide synthesis do not occur when the addition of
a-amanitin is delayed for 6 h suggests that changes in the synthesis of certain
polypeptides during maturation are dependent upon a longer period of transcriptional activity but that the resumption of meiosis is dependent upon RNAs
synthesized at the beginning of maturation.
Finally, it is well documented that the process of meiotic maturation is accompanied 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 claimed
that this reprogramming of protein synthesis is dependent upon the mixing of the
oocytes 'nucleoplasm' and cytoplasm resulting in the mobilization of preformed
mRNAs stored in the cytoplasm (Schultz & Wassarman, I977a,b; Schultz et al.
1978). While a causal relationship between these two events has still to be determined, our experiments show that both GVBD and the changes in protein
synthesis which characterize maturation in sheep oocytes are dependent upon an
initial transcriptional event at the onset of maturation which precedes GVBD.
We are therefore unable to support the view that RNA synthesis is not necessary
for the resumption of meiosis in mammalian oocytes.
We thank Mr Ian Crosby for his skilled technical assistance and Dr D. E. Walters of the
A.R.C. Statistical Group, Department of Applied Biology, Cambridge for his advice on the
statistical analysis. The purified gonadotrophin used in this study was generously donated by
the National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of
Health, Bethesda, Maryland. One of us (J. C. Osborn) is indebted to the Medical Research
Council for financial support.
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{Accepted 26 July 1982)