/. Embryol. exp. Morph. Vol. 43, pp. 157-166, 197S
Printed in Great Britain © Company of Biologists Limited 1978
1 57
Parthenogenetic activation of mouse oocytes induced
by inhibitors of protein synthesis
By G. SIRACUSA, 1 D. G. W H I T T I N G H A M , 2 M. M O L I N A R O 1
AND E. V I V A R E L L P
From the University of Rome and the MRC Mammalian Development
University College, London
Unit,
SUMMARY
Recently ovulated mouse oocytes at the Metaphase II stage undergo parthenogenetic
activation (as indicated by the formation of pronuclei) when incubated for 6 h in the presence
of cycloheximide or puromycin; the activation response increases progressively with the
concentration of inhibitor. Activation is induced with concentrations of cycloheximide that
depress protein synthesis by more than 70 %. Pronoculear formation occurs when protein
synthesis is almost totally inhibited. Incubation of oocyte in Actinomycin D failed to initiate
activation. The results show that the Metaphase II oocyte of the mouse synthesizes protein
factor(s) which are necessary for the maintenance of the meiotic block. Other protein(s)
having opposite effects and a different rate of turnover may also participate in activation
since when the oocytes are treated with a high concentration of cycloheximide (10 fig ml-1)
for varying periods of time, or with varying concentrations for a short period of time (1 h),
a more complex activation response curve is obtained.
Oocytes activated with cycloheximide are capable of further development, following
transfer to the oviducts of pseudopregnant recipients, in a proportion similar to that of
oocytes activated in other ways.
INTRODUCTION
The mechanisms responsible for the pre-fertilization meiotic block, occurring
in the oocyte of most mammals at the second metaphase stage, are unknown.
Studies with frog oocytes suggest that Metaphase II-arrest results from the
production of a cytostatic factor, as yet unidentified, in the cytoplasm of the
secondary oocyte, and this factor disappears following fertilization or artificial
activation (Masui & Markert, 1971). When the oocytes of the marine invertebrate Chaetopterus are treated with cycloheximide, meiosis is resumed and the
oocytes proceed from Metaphase I to the emission of the second polar body
(Zampetti-Bosseler, Huez & Brächet, 1973). Artemia salina eggs remain at the
Metaphase I stage of meiosis following sperm penetration when they are removed
prematurely from the uterus but if treated on removal with low concentrations
1
Author's address: Université di Roma, Istituto di Istologia ed Embriologia Generale,
Via Alfonso Borelli 50, 00161 Roma, Italy.
2
Author's address: MRC Mammalian Development Unit, Wolfson House, University
College London, 4 Stephenson Way, London, NW1 2HE, England.
H
E M B 43
158
G. SIRACUSA AND OTHERS
of cycloheximide (~ 10~7 M) normal development ensues (Fautrez & FautrezFirlefyn, 1974). The results presented in this paper indicate that protein synthesis
is necessary for the maintenance of the mei otic block at Metaphase II in mouse
oocytes since they will complete meiosis and develop parthenogenetically when
protein synthesis is inhibited.
MATERIALS AND METHODS
Collection and culture of oocytes
Oocytes were released from the oviducts of superovulated 6-8 week old
CD1 mice (Charles River, Italy) at various times after the injection of human
chorionic gonadotrophin (HCG) (Edwards & Gates, 1959). The oocytes and
surrounding cumulus cells were incubated in a mouse embryo culture medium
(Whittingham, 1971) supplemented with 10 mM HEPES buffer and the required
concentration of cycloheximide (Calbiochem). The oocytes from the right and
left oviducts of each female were allotted to different experimental treatments in
in order to minimize biological variation. After varying periods of incubation
in cycloheximide, the oocytes were washed free of inhibitor and then cultured
in the standard embryo culture medium. The total period of incubation including
exposure to cycloheximide was always 6 h. All incubations were carried out at
37 °C in 5 ml test-tubes gassed with a mixture of 5 % 0 2 , 5 % C0 2 and 90 % N 2 .
Following incubation the oocytes were treated with hyaluronidase and washed
in tissue culture medium to remove the cumulus cells. Degenerated oocytes
were discarded and the remaining oocytes were fixed in 10 % buffered formalin
before they were examined for activation with Nomarski optics at 500 x magn.
Oocytes were scored as activated if pronuclear formation had occurred.
Oocytes removed 1 5 | h after the injection of HCG were also treated with
varying concentrations of puromycin (Calbiochem) or Actinomycin D (Serva)
for 6 h and then examined for signs of activation.
Protein synthesis
To assay protein synthetic activity in oocytes exposed to cycloheximide,
oocytes were cultured for 1 h in a medium containing varying concentrations of
cycloheximide and tritiated leucine ([4,5-3H]L-leucine; 40/^Ci ml -1 , specific
activity 60 Ci mmol -1 , New England Nuclear). After incubation they were
washed five times in culture medium containing cold leucine. Samples of
15 oocytes were disrupted by freezing and thawing three times and 100 /ig BSA
was added to each sample as a carrier protein to facilitate the recovery of the
oocyte proteins following precipitation with 10 % cold trichloroacetic acid
(TCA). The precipitate was washed three times with 5 % TCA (100 /d/wash),
dissolved in 20/d of 0-8 N - K O H and counted in a toluene based scintillation
fluid containing 2 % Biosolv (Beekman). Background values were obtained by
counting aliquots of the fluid from the final wash. At each cycloheximide concentration three to four samples of oocytes were prepared and counted.
Egg activation by inhibitors of protein synthesis
159
100 r
I
0
J L
I
' ' 00001
I
I
l
0001
001
01
Cycloheximide concentration (Mg nil ')
1
1
1
10
Fig. 1. The percentage of parthenogenetically activated mouse oocytes collected at
15^ h post-HCG and exposed for 6 h to various concentrations of cycloheximide.
Percentage activation ranges are shown for concentrations tested in more than one
experiment.
Table 1. Parthenogenetic activation of mouse oocytes* induced by treatment
with puromycin for 6 h
Puromycin
concentration
(/*g/ml-1)
Activation
(%)
No. of eggs
examined
00
1-2
(162)
1
5
10
5-2
48 0
59-9
(58)
(50)
(222)
15
541
(74)
20
461
(91)
* Oocytes removed 15^-h post-HCG.
Transfer of activated oocytes
Oocytes from C57BL and Fx (C57BL x A2G) hybrid females were activated
by treatment with 5-10/*g-1 cycloheximide for 6 h and transferred to the
oviducts of pseudopregnant recipients either as pronucleate oocytes immediately
following incubation in the inhibitor or as 2-cell ova following a further 24 h
culture in standard medium. The recipients were either MF1 (Olac) or F x
(C57BL x CBA)hybrid females that were mated with sterile males (carrying the
T145H translocation, Lyon & Meredith, 1966). Both stages were transferred on
the first day of pseudo-pregnancy, i.e. the day on which the vaginal plug was
found and the females were killed and examined for implantation sites 6 days
later (the seventh day of pseudopregnancy). All implantation sites were fixed
and prepared for histological examination.
II-2
160
G. SIRACUSA AND OTHERS
'.«,
' f *•*;,• ^ » ^ '
Fig. 2. The types of parthenogenones induced by treatment with cycloheximide:
(A) One pronucleus, second polar body. (B) Immediate cleavage. (C) Two pronuclei,
no second polar body. (D) One pronucleus, no second polar body. Nomarski optics.
RESULTS
Recently ovulated mouse oocytes at the Metaphase II stage (15-^ h post-HCG)
undergo parthenogenetic activation as indicated by the formation of pronuclei
when incubated for 6 h in the presence of cycloheximide (Fig. 1). The results of
two experiments are combined in thefigure;an overall total of 934 oocytes were
observed (53-220 oocytes at each concentration). The percentage of activated
oocytes increased with the concentration of cycloheximide. Activation was also
Egg activation by inhibitors of protein
synthesis
161
100
50 -
J
001
01
1
L
10
100
Cycloht'ximide concentration (/ig nil*1)
Fig. 3. The percentage of mouse oocytes activated following incubation in varying
concentrations of cycloheximide for 1 h ( O — O ) compared with the percentage
inhibition of protein synthesis occurring in similarly treated oocytes ( # — # ) .
Percentage activation ranges are shown for concentrations tested in more than one
experiment.
induced by treatment of oocytes for 6 h with varying concentrations of puromycin (Table 1) but the activation response declined with exposure to concentrations higher than 10 /tg ml -1 . Examples of the four major types of
parthenogenones (Tarkowski, 1975) were produced (Fig. 2) although the group
with one pronucleus and second polar body was predominant, e.g. out of
74 oocytes activated in the presence of 10 jug ml - 1 cycloheximide, 66 had one
pronucleus and the second polar body, two had immediately cleaved (Braden
& Austin, 1954), two had one pronucleus and no second polar body and four
had two pronuclei and no second polar body. The morphological appearance of
the activated oocytes was normal, the pronuclei were well formed and contained
one or more nucleoli. Incubation of oocytes with Actinomycin D (0-05 and
0-1 fig ml -1 ) failed to initiate activation above control levels.
Tn the next series of experiments Metaphase II oocytes at 15^ h post-HCG
were treated with various concentrations of cycloheximide for 1 h, followed by
culture in a standard medium for a further 5 h. The results of two experiments
are combined in Fig. 3; an overall total of 1099 oocytes were examined (79-190
oocytes at each concentration). The dose-response curve was found to be more
complex than the activation response observed after prolonged incubation in
cycloheximide (Fig. 1); a peak of activation was obtained at 1 [igm\~l cycloheximide followed by a second rise in activation at concentrations greater than
3/6gml_1. When the response is compared with the incorporation of [3H]leucine in
the presence of inhibitor,oocytes are activated by concentrations of cycloheximide
that depress protein synthesis by more than 70 %. No significant differences
162
G. SIRACUSA AND OTHERS
Cycloheximide treatment (h)
Fig. 4. The effect of treating mouse oocytes at various post-ovulatory ages with
a fixed concentration of cycloheximide (10 fig ml-1) for different periods of time.
a, b and c represent the percentage activation of oocytes collected at 14, 15£ and
16|h post-HCG respectively. Percentage activation ranges are shown for times
tested in more than one experiment.
were found in the rate of decay of the proteins synthesized in the presence of
various concentrations of cycloheximide (0-01-10 fig ml-1) and it was also
found that oocytes were incorporating levels of labelled leucine similar to the
untreated controls within 20 min of removal from 10 jug ml -1 cycloheximide
(unpublished observations).
A qualitatively similar response was obtained when oocytes were treated with
a high concentration of cycloheximide (10 fig ml-1) for varying periods of time
(Fig. Aa-c) instead of treating oocytes for a fixed period of time in varying
concentrations of the inhibitor. The results of 2-3 experiments are combined in
the figure; overall totals of 554, 890 and 950 oocytes were examined at 14, 15+
Egg activation by inhibitors of protein synthesis
163
100 r-
-
50
01-
_1
2
I
3
l_
4
Cyclohcximide treatment (h)
Fig. 5. The effect of treating mouse oocytes at 15-^ h post-HCG with a low concentration of cycloheximide (0-3/tgml-1) for varying periods of time. Percentage
activation ranges are shown for times tested in more than one experiment.
and 16^ h post-HCG respectively (48-190 oocytes at each exposure time). The
initial activation peak occurs after a period of exposure to the inhibitor which
becomes progressively shorter with the increase in post-ovulatory age of the
oocyte (in GDI females ovulation is completed by approx. 12 h after the
injection of HCG, unpublished observations). If lower concentrations of
inhibitor are used (0-3 /-eg ml -1 ) the initial peak of activation is not discernible
(Fig. 5). Three experiments are combined in the figure; an overall total of
1243 oocytes were examined (86-220 oocytes at each exposure time). The
response reaches a maximum after 3-4 h exposure after which there is a slight
decline.
Oocytes activated with cycloheximide were capable of further development
following transfer to the oviducts of pseudopregnant recipients (Table 2).
Although the number of oocytes transferred was comparatively small, the
proportion of activated oocytes developing to the stage capable of initiating
a decidual response was similar to the development of oocytes activated in
other ways (see reviews by Graham, 1974; Tarkowski, 1975). The decidual
cell reaction appeared normal but giant cells and embryonic tissue were only
discernible in a small proportion of the implantation sites. In the few instances
where the implantation sites contained embryos, embryonic development was
retarded by approximately 24 h. Haploid embryos originating from inbred
rather than hybrid mice did not appear to have a greater development potential
as suggested previously (Kaufman, Huberman & Sachs, 1975). The larger
number of implantation sites resulting from the transfer of activated oocytes
after culture to the 2-cell stage (13/21-62 %) may be due to the extra time the
164
G. S I R A C U S A A N D O T H E R S
Table 2. Development of mouse oocytes activated by cycloheximide treatment
(5-10 jag ml*1 for 6 h) following transfer to the oviducts of recipients on the first
day of pseudopregnancy
Source of oocytes*
Stage at transfer
No. of
oocytes
transferred
to $$ with
implantation
sites
Total
no. of
implan- No. of
tation
implansites on
tation
Day 7 sites with
of
visible
pseudogiant
pregnancy cells
No. of
implantation
sites with
embryonic
tissue
1-cell
40
10
4
2
(1 pronucleus)
Fi (C57BL x A2G)
1-cell
54
9
3
1
(1 pronucleus)
Fi (C57BL x A2G)
2-cellf
21
13
7
1
* Oocytes removed from $$ between 15 and 16 h following the injection of HCG.
t 21/22 activated oocytes with 1 pronucleus developed to the 2-cell stage after 24 h in
culture before transfer.
C57BL
embryos spent in utero before implantation, for haploid blastocysts have
significantly lower cell numbers when compared with diploid controls (fertilized)
of similar developmental age due to a slower cleavage rate after the 4-cell stage
(Kaufman & Sachs, 1976).
DISCUSSION
Our results show that the Metaphase II oocyte of the mouse synthesizes
protein factor(s) which are necessary for the maintenance of the meiotic block.
A similar phenomenon may be responsible for the meiotic blocks occurring in
the oocytes of other classes of animals (Masui & Markert, 1971; ZampettiBosseler et al. 1973; Fautrez & Fautrez-Firlefyn, 1974). The formation of
pronuclei in the presence of cycloheximide or puromycin indicates that this
process is not dependent upon the synthesis of new proteins and the inability of
Actinomycin D to activate the oocytes suggests that the synthesis of the
inhibitory protein(s) is not controlled at the transcriptional level. The treatment
of oocytes with cycloheximide for 6 h does not impair their ability to develop at
least to the stage of implantation.
'Activating' effects of various types can be induced in other repressed cellular
systems by treatment with inhibitors of protein synthesis. In serum-starved and
density-inhibited human fibroblasts cycloheximide causes an increase in
putrescin transport which is a characteristic response associated with the
initiation of cell proliferation (Pohjanpelto, 1976). Uridine uptake is increased
in serum-deprived mouse fibroblasts treated with cycloheximide (Hershko,
Egg activation by inhibitors of protein synthesis
165
Mamont, Shields & Tomkins, 1971) and RNA synthesis is stimulated in aminoacid-deprived HeLa cells treated with cycloheximide (Smulson & Thomas, 1969).
These activating effects are usually attributed to the inhibition of the synthesis
of rapidly metabolising repressor proteins.
The activation of the oocytes may not be due simply to the removal of
'blocking' agents since with short exposures to cycloheximide the activation
response does not increase progressively with increasing concentrations of
inhibitor. From our data, it would appear that the meiotic block and subsequent activation are regulated not only by 'blocking' protein factor(s) but
also by other protein(s) having opposite effects and different rates of synthesis
or decay. Thus, the response obtained with a low concentration of cycloheximide
(Fig. 5) might indicate that under these conditions the intracellular concentration
of blocking factors is affected more than the factors necessary for activation.
The ratio between the 2 factors probably varies during the natural ageing of the
oocyte during the postovulatory period, since there is an increase in spontaneous
activation with age (Austin, 1961 ; Longo, 1974), and an earlier occurrence of
the initial activation peak in the presence of inhibitor with advancing postovulatory age (Fig. 4a-c). Furthermore, the fertilizability of ovulated mouse
eggs also varies with time in a way parallel to activation with cycloheximide:
a dramatic increase occurs between 13 and 15 h post-HCG, and maximal
fertilizability is reached at 17 h (Iwamatsu & Chang, 1971).
We thank Mr M. Coletta and Mrs J. Keogh for technical assistance. This work was
supported by the World Health Organization, the Ford Foundation, the Consiglio Nazionale
delle Ricerche (Biology of Reproduction Grant no. 7600300.85) and NATO.
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{Received 28 April 1977, revised 4 July 1977)