J. Embryol. exp. Morph. Vol. 53, pp. 145-162, 1979
Printed in Great Britain © Company of Biologists Limited 1979
The utilization of an inhibitor of
spermidine and spermine synthesis as a tool for the
study of the determination of cavitation in
the preimplantation mouse embryo
By H. ALEXANDRE 1
From the Departement de Biologie moleculaire,
Universite libre de Bruxelles, Belgium
SUMMARY
The inhibition of spermidine and spermine synthesis by methylglyoxal-Bis(guanylhydrazone) (MeGAG) at concentrations of 5, 10 and 20 /iu, induces a reversible metabolic
quiescence of mouse embryos, cultured in vitro from the 2-cell stage, at an average of 10-2,
8-5 and 6-9 cell stages respectively. In contrast, the inhibition of putrescine synthesis by
<x-methylornithine (a-MeOrn) at concentrations up to 10 mM fails to inhibit blastocyst
formation, as shown previously.
Complete reversibility of this induced arrest of development is observed for treatments
up to 31 h with MeGAG at 10 JAM. In agreement with the biological clock theory of Smith
& MacLaren's hypothesis, the delay in cavitation is proportional to the length of treatment.
However, the average cell numbers of the 'delayed nascent blastocysts' of all treated
embryos (21-8-24-2) are consistently lower than that of control embryos (33-6) irrespective
of the duration of treatment. It seems therefore that under some experimental conditions,
DNA and chromosome replication on the one hand and cytoplasmic maturation on the
other may be dssynchronized. This suggests a role for a cytoplasmic factor in the induction
of cavitation.
INTRODUCTION
The earliest process of cellular determination during mammalian development
is the progressive establishment of two distinct populations of cells in the latemorula to early-blastocyst stage: the trophectoderm and the inner cell mass
(ICM). These two cell types clearly differ from each other in a number of
ways which include: cell shape, embryological fate (Gardner, 1971, 1972),
[3H]thymidine-labelling indices (Barlow, Owen & Graham, 1972), mitotic rate
(Copp, 1978), junctional complexes (Nadijcka & Hillman, 1974; Ducibella,
Albertini, Anderson & Biggers, 1975), phosphatase activities (Mulnard, 1974;
Mulnard & Huygens, 1978; Vorbrodt, Konwinski, Solter & Koprowski, 1977;
Johnson, Calarco & Siebert, 1977; Izquierdo & Marticorena, 1975), esterases
isoenzyme (Sherman, 1972), cell surface properties (Calarco & Epstein, 1973),
1
Author's address: Laboratoire de Cytologie et Embryologie moleculaires, Departement
de Biologie moleculaire, U.L.B. 67, rue des Chevaux, B-1640 Rhode-Saint-Genese, Belgium.
146
H. ALEXANDRE
Ornithine
SAM
1
p
*- dcSAM
*• Putrescine
<________^ Spermidine
MeGAG
Spennine
Fig. 1. Sites of inhibition of polyamine synthesis by a-MeOrn and MeGAG. SAM,
S-adenosylmethionine; dcSAM, decarboxylated SAM.
susceptibility to both infective and oncogenic viruses (Glass et al. 1974;
Abramczuk, Vorbrodt, Solter & Koprowski, 1978), and synthetic patterns of
polypeptides (Handyside & Johnson, 1978).
Two main theories have been proposed to explain the determination of the
trophectoderm versus that of the ICM. The first, which is mainly based on
histochemical data postulates the existence of a cytoplasmic heterogeneity
which confers 'trophoblastic properties' to some blastomeres during early
cleavage whereas the other blastomeres are predetermined as ICM precursors
(Dalcq & Seaton-Jones, 1949; Mulnard, 1955; Seidel, 1960; Dalcq, 1966). The
second theory which is based on the experimental data made available by the
use of the in vitro culture of preimplantation stages, supports the idea that
determination depends on the position of the blastomeres in the morula
(Tarkowski & Wroblewska, 1967) such that inner cells become ICM whereas
outer cells become trophectoderm. This is the well known 'outside-inside
model' (Herbert & Graham, 1974), which is almost generally accepted now,
at least for the mouse. The problem of the determination of the trophectoderm
and the ICM has been reviewed in detail by Mulnard (1966), Herbert & Graham
(1974) and Denker (1976).
The formation of the blastocoele in the mouse has been described extensively
by Calarco & Brown (1969). It results from the confluence of intercellular
spaces progressively filled with fluid released from cytoplasmic vesicles whose
number and size have increased subsequent to fertilization. However, little
is known about the actual trigger for the appearance of the blastocoele. The
work of Tarkowski & Wroblewska (1967), on the development of isolated
blastomeres from 4-cell and 8-cell stages, has led to the conclusion that, irrespective of the cell number, cavitation occurs at a precise time after fertilization. More recently, the use of cytochalasin B enabled Smith & McLaren
(1977) to conclude that an important factor seems to be either the number of
chromosomal divisions or DNA replications after fertilization, or the nucleocytoplasmic ratio reached at that time. The use of suitable reversible inhibitors
may therefore provide useful models for these studies.
It is now clearly established that polyamines (putrescine, spermidine and
spermine) play an essential role in cellular metabolism and proliferation (see
Polyamines synthesis and blastocyst formation
147
Janne, Poso & Raina, 1978). Recently, selective inhibitors of polyamines biosynthesis have become available, among which are (i) a-methylornithine (aMeOrn), a specific inhibitor of ornithine decarboxylase and (ii) methyl-glyoxalBis(guanylhydrazone) (MeGAG), an inhibitor of S-adenosyl methionine decarboxylase (Fig. 1 indicates the precise sites of action of the two inhibitors).
Both a-MeOrn (Mamont et al. 1976) and MeGAG (Heby, Marton, Wilson &
Gray, 1977) selectively inhibit DNA replication and cell proliferation. In contrast
to sea urchin (Brachet et al. 1978) and Polychaete eggs (Emanuelsson & Heby,
1978), preimplantation mouse embryos are much more affected by MeGAG
than by a-MeOrn (Alexandre, 1978a). Moreover, inhibition of spermidine and
spermine accumulation apparently induces quiescence in the embryos at the
8- to 16-cell stage and the resumption of their development after transfer to
fresh medium is followed by a delay in cavitation. The present work has been
performed in order to analyse in more detail the effects of MeGAG on cleavage
and blastocyst formation in relation to the determination of cavitation.
MATERIALS AND METHODS
All the embryos used were obtained from 6 to 10-week-old virgin randombred albino females induced to superovulate by intraperitoneal injections of
5i.u. PMSG (Gestyl: Organon) at 17.00-18.00 h followed by 5 i.u. hCG
(Pregnyl: Organon) 48 h later. They were then caged with males overnight.
Fertilization was assumed to occur at 06.00 h on the following day and was
therefore considered as time 0 of embryonic development. On Day 2 of
pregnancy, the embryos were removed by flushing the oviducts and the 2-cell
stages were placed in organ culture dishes (Falcon Plastics) in 50 jul drops of
Whittingham's culture medium (Whittingham, 1971), under paraffin oil, and
incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air.
MeGAG was purchased from Aldrich Chemical Company Inc., Milwaukee,
Wisconsin, U.S.A. It was dissolved in the culture medium at concentrations
varying from 1-25 to 20 JU,M.
The effect of MeGAG on RNA synthesis has been tested by autoradiography,
after incorporation of 5-[3H]uridine 25 Ci/mM (Radiochemical Centre, Amersham, England). The medium which contained 10/tCi/ml was prepared as
described elsewhere (Alexandre, 1977). Some preparations were incubated
with either 0-1 mg/ml bovine pancreatic RNAase in Tris-buffer pH 7-4 or
0-2 mg/ml DNAase in Tris-buffer MgCl2 M/300 pH 7-4, for 2 h at 37 °C. All
slides were treated with 2 % perchloric acid for 30 min at 4 °C and rinsed
overnight in tap water. They were autoradiographed with Ilford emulsion L4,
using the dipping method, and exposed for 5 days.
The embryos were prepared for cell counting and for autoradiography by
the air-drying method (Tarkowski, 1966) and were stained with Giemsa.
To determine cell death within the preimplantation embryos, a 0-005 %
148
H. ALEXANDRE
Fig. 2. 72 h old embryos cultured from the 2-cell stage in control medium (A) or
in 10/*M MeGAG containing medium (B).
solution of eosin yellow (Merck, AG Darmstadt) in phosphate-buffered saline
(Oxoid, London) was used as described by Bellve (1973). Since eosin Y
exclusion by cells is believed to be dependent on the normal functioning of the
cell membrane (Goldstein & Okada, 1969), blastomeres which do not exclude
the strain may be considered as dead. The embryos were examined for stained
blastomeres under a Wild M20 microscope (100 x ) between 2 and lOmin
after staining.
Nascent blastocysts were defined using the same criteria as Smith & McLaren
(1977), i.e. the presence of one or more intercellular spaces when seen under
the dissecting microscope.
RESULTS
Effect of continuous treatment with MeGAG on blastocyst formation
Mouse eggs were cultured in vitro from the 2-cell stage for about 65 h either
in control medium or in media containing various concentrations of MeGAG.
At that time, the majority of the embryos had reached the full grown blastocyst
stage and some of them had hatched.
As judged by the percentage of blastocysts (Table 1 and Fig. 2), relatively
low doses of MeGAG exert a dramatic effect on cavitation such that at 10 /*M
or more, MeGAG induces an almost total arrest of development before cavitation while at 5 /JM only about 10 % of the embryos cavitate.
The few treated embryos which developed until the blastocyst stage cavitated
somewhat later than the controls. For instance, in expt 2, about half of the
Polyamines synthesis and blastocyst formation
149
Table 1. Effect of MeGAG 5, 10 and 20 /AM on blastocyst formation in vitro.
Embryos were cultured from the 2-cell stage for 65 h {100 h-old embryos)
Developed embryos
Treatment
No. of
<M MeGAG) embryos
Expt 1
0
5
10
20
0
5
10
20
Expt 2
Uncleaved
2-cell eggs
60
70
70
72
75
79
80
80
4
9
4
13
Blastocysts
j\
' Morulae'
Blastocysts
10
54
64
56
13
71
73
77
46
7
2*
3*
61
7
2*
0
77
10
3
4
81
9
3
0
Very small vesicular forms.
Table 2. Effect of low doses of MeGAG on in vitro development of mouse eggs:
the mean cell number of arrested morulae and blastocysts cultured from the
2-cell stage for 65 h (100 h-old embryos)
Developed embryos
'Morulae'
Treatment
Uncleaved
(/*M
No. of 2-cell
MeGAG) embryos eggs
No.
0
1-25
2-5
5
48
59
59
60
2
11
8
8
17
22
26
46
Blastocysts
Mean cell no.
(±S.E.)
Range
No.
Mean cell no.
(±S.E.)
Range
8-8±l-3
71+0-9
9-2 ± 1 0
7-1 ±0-5
3-21
3-21
4-18
3-14
29 (60 %)
26 (44 %)
25 (42 %)
6(10%)
43-9 ±2-3
26-8 ±2-2
210±l-8
120±2-6
16-82
6-48
9-46
6-22
final number of the blastocysts were already formed in the control group after
48 h of culture whereas no cavitation had occurred in the treated groups.
Moreover, in contrast to the untreated controls, the ' small blastocysts' formed
from the 2-cell eggs incubated in the presence of MeGAG never hatched
in vitro even when left in culture for an additional period of time. Since it is
generally believed that in vitro hatching is a mechanical process that depends
on the number of cells in the blastocyst, cell counts were made on embryos
treated with MeGAG at concentrations of 5 (iu or less, which should allow
more than 10 % of the embryos to cavitate. The cell counts were performed
separately on uncavitated and cavitated embryos and are recorded as 'morulae'
and 'blastocysts' respectively in Table 2.
While, in this experiment, an abnormally low percentage (60 %) of blastocysts was obtained, the mean cell number of treated blastocysts was significantly
lower than that of the controls (43-9, this value is of the same order of magnitude
150
H. ALEXANDRE
as that found by Smith & McLaren, 1977). An excellent correlation is observed
between MeGAG concentration, blastocyst number and the mean cell number
of the blastocysts. The presence of vesicular forms with only six and nine
cells, recorded as ' blastocysts' in Tables 1 and 2, clearly indicates the existence
of abnormal forms (mainly in the 5 fiu group) which correspond to the false
blastocysts, the trophoblastic vesicles and the non-integrated forms exhaustively
described by Tarkowski & Wroblewska (1967). On the other hand, it is
interesting to note that the MeGAG-blocked embryos recorded in Tables 1
and 2 looked healthy until the end of the culture (3 days) and did not show
any signs of degenerative processes (Fig. 2). This point has been confirmed
by staining with eosin Y. Samples of 25-40 control embryos or embryos
treated with MeGAG at 10 /AM were transferred to PBS containing 0-005 %
eosin Y, at various times after the beginning of treatment (21, 41 and 65 h)
and were observed 2 min later for cell death. Their development is shown
in Table 3.
Of all the embryos analysed for cell death (see Table 3), only one control
blastocyst (an embryo cultured for 65 h) contained any dead blastomeres.
Surprisingly none of the treated embryos, even after exposure to the inhibitor
for 65 h, contained dead blastomeres. Moreover, the eggs arrested at the 2cell stage were still able to exclude the stain. Therefore, it can be concluded
that MeGAG induces a metabolic quiescence without killing the embryos.
Since it is very difficult, with the dissecting microscope, to determine the
precise number of cells in living embryos with more than six to eight cells,
air-dried preparations of control and treated embryos (5,10 and 20 f.iu MeGAG)
were examined (Table 4) in the same way as shown in Table 2 for lower concentrations. This enabled us to determine the precise stage at which the
embryos were arrested and which were recorded as 'morulae' in Tables 1 and
3.2-cell and a few 4-cell-stage embryos were cultured for 65 and 45 h respectively
and fixed on slides. It is clear from Table 4 that the treated embryos were
blocked at an early stage of cleavage and that at 5 jtm, MeGAG provoked the
arrest of development at the early fourth cell cycle (embryos optimally arrested
at the 9- to 12-cell stage), whereas at 10 and 20/*M, the arrest occurred during the
third cell cycle. No significant difference was observed between the 2- and 4-cell
stages when treated with 20 fiu MeGAG. In agreement with the results presented
in Table 2, the few treated embryos (mainly with 5 JLLM) which cavitated were
those that contained the highest cell number.
When a similar analysis is performed at the beginning of the treatment, i.e.
during the first cell cycles, it seems that 10 and 20 JUM. MeGAG slows down
the mitotic rate very early during cleavage (Table 5).
From this experiment and from Table 4, it is evident that 10 /IM MeGAG
does not immediately interfere with cleavage since the embryos apparently
progress normally through the second cell cycle. However, after the third cell
cycle, they are considerably slowed down and they only reach an average cell
0
10
0
10
0
10
40
40
25
25
20
19
No. of
embryos
1
5
4
6
2-cell
—
2
7
—
3-cell
1
25
21
2
3
1
2
1
1
1
5-cell
4-cell
Stages
3
1
4
1
2
6-cell
7
2
21
14
5
12
'Morulae'
0
5
10
20
0
20
OM
NO.
75
76
79
76
23
23
of
MeGAG) embryos
Treatment
6
7
17
14
—
4
2-4
3
17
42
46
—
10
5-?
3
36
17
14
.—
8
9-12
3§
2
—
1
14t
1
13-16
17-20
21-24
No. of embryos according to their no. of cells
23*
61*
30
46-lf±2-6
10-2±M
8-5 ±0-9
6-9 ±0-8
45-6||±2-0
81 ±0-7
Mean cell no.
±S.E.
2
24
65
8
Total
no. of
mitoses
The numbers of blastocysts as percentages of total embryos cultured from the 2-cell stages are in total agreement with
those presented in Table 1.
* Embryos having cavitated.
t Range: 31-98.
% Among which five embryos have cavitated.
§ Among which two embryos have cavitated.
|| Range: 32-64.
4-cell
2-cell
Stage
at start of
treatment
14
—
—
Blastocysts
Table 4. Determination of the stage of arrest of development induced by MeGAG. 2- and 4-cell-stage embryos
were cultured for 65 h and 45 h respectively
65
41
21
Time after the
beginning of
Treatment
treatment
(/*M MeGAG)
(h)
Table 3. Distribution of control and MeGAG-treated embryos, prepared for analysis of cell death, according to
their stage, at three different times after the beginning of the culture
5
O
Co
152
H. ALEXANDRE
Table 5. Effect of 10 or 20 JLIM MeGAG on the first cleavages following
treatment of 2-cell-stage embryos in vitro
Duration
Expt 1
Treatment
of
OM
treatment
MeGAG)
0
10
Expt 2
0
20
Cell
(h)
No. of
embryos
Mean cell no.
±S.E.
22
46
22
46
51
39
53
33
25
42
25
42
25
26
26
25
4-8 + 0-3
17-7±l-6
4-5 ±0-2
6-9 ±0-4
4-7 ±0-4
10-7±0-8
41 ±0-2
5-6 ±0-4
Total no.
no.
of
range
mitoses
2-8
13
13
8
1
3-33
2-8
3-12
3-8
4-16
2-7
2-8
8
20
1
1
Fig. 3. Autoradiographs of three different 8-cell-stage embryos, cultured for 4 h
in the presence of 5-[3H]uridine. They are representative of the three arbitrary
levels of label, used in Table 6: (A) heavy (+ + +), (B) normal (+ +) and (C)
weak (+).
number of 8-5. The same conclusion can be drawn from experiments with 20 JLLM.
MeGAG where the slowing down effect appears earlier and there are very few
mitoses, indicating that the cellular arrest is induced during interphase.
Thus, since it induces a total inhibition of cavitation with a minimum of
' early effects ',10 /AM MeGAG has been used in all of the experiments performed
in order to induce metabolic quiescence.
To examine whether some metabolic processes are progressively switched
off by 10 /AM MeGAG during the third cell cycle, the incorporation of [3H]-
Polyamines syn thesis and blastocyst formation
153
Table 6. Autoradiographic estimation of [3H]uridine incorporation in cleaving
mouse embryos cultured from the 2-cell stage with or without 10 /*M MeGAG.
The control and treated embryos were fixed immediately after the radioactive
pulse
Incubation
time after start
of culture
(h)
18-22
42-46
Treatment
Control
MeGAG
Control
MeGAG*
No. of
embryos
42
42
34
25*
Incorporation level
(arbitrary classification)
Range
(cell
number)
+++
++
+
2-8
2-8
3-33
4-12
18
24
30
4
14
11
2
2
10
7
2
19
* Only embryos which had reached the 4-cell stage have been recorded because one
2-cell and one 3-cell stage were unlabelled.
uridine has been studied in embryos treated in exactly the same way as those
of expt 1 in Table 5. Embryos were cultured for either 18 h or 42 h in control
or MeGAG containing medium, transferred to the same media containing
5-[3H]uridine for 4 h and then fixed for autoradiography. The embryos were
arbitrarily classified as heavily (+ + +), normally (+ + ) or weakly ( + ) labelled;
this is illustrated in Fig. 3 and summarized in Table 6.
No differences in either cytoplasmic, nuclear or nucleolar labelling could be
detected by autoradiography between control and MeGAG-treated embryos,
when they were incubated from 18 to 22 h after the beginning of treatment. In
contrast with the normal situation however, no label was seen in the few
embryos which were treated with RNAase; this reflects the arrest of DNA
synthesis (Alexandre, 1977). RNA synthesis is markedly decreased after long
(42-46 h) continuous treatments with MeGAG. Inhibition of RNA synthesis
is thus only observed in slowly dividing or arrested embryos where a residual
synthesis remains measurable.
Reversibility of MeGAG effects
In a first group of experiments, embryos were cultured in the presence of
MeGAG from the 2-cell stage, transferred at several selected times to fresh
medium and then analysed for the appearance of the blastocoele.
As can be seen from Table 7, a good reversibility of the MeGAG treatment
was observed, and the arrested embryos have not lost their capacity to cavitate
provided that the treatment did not exceed 21 h. The in vitro hatchability was
however decreased after 21 h treatment by MeGAG at the concentrations
tested. This can again be considered as a result of a lower cell number per
embryo.
It is more interesting that the restoration of cavitation by transfer into
154
H. ALEXANDRE
Table 7. Effect of 10 and 20 /IM MeGAG on blastocyst formation and in vitro
hatching, according to the duration of treatment from the 2-cell stage onward',
embryos were all cultured for 92 h
Duration
of
MeGAG
cone.
10/tM
20 fiwi
frpo tmpnt
11 WAI 111VUI
Blastocysts
"NTn
nf
j
Hatched
blastocysts
No. of
as %of
Ha tr*hpH
total
blastocysts blastocysts
\.\J l a l
(h)
embryos
No.
0
17
21
92
0
70
70
70
15
65
21
48
65
65
49
42
41
—
52
52
92
62
/o
70
60
59
—
80
38
32
19
—
41
78
76
46
—
79
9*
80
14
5*
8
20
—
—
38
—
—
* Very small vesicular forms.
fresh medium is delayed compared with the controls. For instance, in the
experiment with 20 fiu MeGAG (Table 7), in spite of the fact that at the
end of culture, the same numbers of blastocysts were obtained in both groups,
only 7 blastocysts were seen in the group treated for 21 h, while 21 blastocysts
had already formed in the control group 54 h after the beginning of culture.
Thus, although autoradiography suggests that RNA synthesis is not affected,
MeGAG seems to induce a metabolic quiescence with regard to DNA replication
and cellular proliferation.
To confirm the existence of a delay induced by transitory quiescence, a
systematic examination of embryos treated from the 2-cell stage with 10 /iu
MeGAG for different lengths of time was undertaken, using a dissecting microscope to score the formation of blastocyst.
Figure 4 shows that MeGAG given at the beginning of the culture induces
a delay in cavitation which is proportional to the duration of treatment. When
the blastocysts which are formed during one additional day are added to the
values obtained at the end of the systematic examinations (104 h-old embryos
on Fig. 4), it can be seen that an excellent reversion is obtainable for treatments
up to 31 h whereas a 40 h-long treatment is almost totally irreversible (Table 8).
In expt 2 of Table 8, nascent blastocysts were recovered every 1 h 30 min
and immediately fixed on slides, in order to estimate their mean cell number in
relation to the duration of the treatment and the delay recorded on Fig. 4B.
It should be noted, from Table 9, that the mean cell number of 'delayed
nascent blastocysts' is significantly lower (about 10 cells less) than that of
controls (P < 0-01); it is of the same order of magnitude for all treated
embryos, irrespective of the duration of treatment except for long periods of
Polyamines synthesis and blastocysi formation
155
A
30 -
20-
10-
76 77
80 83 86 89 92 95 98 101 104
50 A
2
40-
30-
20-
10 -
76 77
80
83
86 89 92 95 98
Age (h after fertilization)
101 10*
Fig. 4. Effect of 10 /AM MeGAG on the appearance of the blastocoele, according
to the duration of treatment, before transfer to fresh medium: 0 h, A
A; 16 h,
O
O; 21 h, •
B ; 2 6 h , • — • ; 36h, A
A;40h, •
Q.
(A) Experiment 1. (B) Experiment 2. The embryos were cultured from the 2-cell
stage; in both experiments, the first control blastocysts were recorded at 42 h after
the beginning of culture while the last observations were made at 69 h and 70 h
30 min after the beginning of culture in expt 1 and 2 respectively.
156
H. ALEXANDRE
Table 8. Effect of 10 JUM MeGAG on final blastocyst formation, according to the
duration of treatment: same experiments as those recorded in Fig. 4 A (expt 1)
and B {expt 2), except that the embryos were cultured for 92 h
Blastocysts
T"\-» * **Q \ i r\
Hatched blastocysts
n
A
l/UlaUOn
As%
of tota 1
embryos
of
treatment
(h)
No. of
embryos
0
16
21
26
31
40
0
45
45
45
45
45
45
60
44
78
71
71
51
53
16*
73 ^
16
60
51
85
21
26
31
40
60
60
60
60
49
43
42
18*
82
72
70
Expt 1
Expt 2
No.
35
32
32
23
24
7*
No.
As%
of total
blastocysts
30
18
13
5
7
1
86
56
40
22
29
14
Not analysed
30*J
* Small vesicular forms.
Table 9. Mean cell number of the nascent blastocysts recovered
during expt 2 (Fig. 4)
Duration of
treatment with
MeGAG 10/*M
(h)
0
16
21
26
31
40
No. of
Mean cell
nascent blastocysts no. of nascent blastocysts
(±S.E.)
analysed
40
47
44
31
25
10
33-6±l-3
24-2±l-l
24-3 ±1-0
231 ±1-3
21-8± 1-3
15-0±M
time (40 h) where we already know that the arrest is irreversible (Table 8). In
the latter case, strong cytological aberrations such as chromosomal fragmentation and micronucleation were seen. Such aberrations have also been
found in some fixed nascent blastocysts derived from embryos treated from
the 2-cell stage for 26 and 31 h but only in the most retarded ones.
Polyamines synthesis and blastocyst formation
157
DISCUSSION
Polyamine synthesis
In all of the biological systems studied to date (i.e. cultured cells, growing
tumours, regenerating liver, kidney or cardiac hypertrophy, etc.), an initial
step in proliferation consists of an increase in the activity of the enzymes
involved in polyamine biosynthesis (see Janne et al. 1978, for review). Similarly,
increases in the activities of both ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) as well as in the level of polyamines
have been recorded during the early embryonic development of Amphibians
(Russell, 1971), sea urchins (Manen & Russell, 1973; Kusunoki & Yasumasu,
1976) and the nudibranch mollusc Phestilla (Manen, Hadfield & Russell, 1977).
However, due mainly to the scarcity of embryonic material, no direct information
about changes in either enzyme activity or polyamine content at fertilization
and during early development in Mammals is available. In the present work,
indirect evidence strongly suggests that polyamines play a key role in genetic
activity during the preimplantation development of the mouse.
In Polychaetes (Emanuelsson & Heby, 1978) and Echinoderms (Brachet et
al. 1978), the inhibition of putrescine synthesis by a-MeOrn from the onset of
development leads to an arrest at the blastula stage whereas treatment with
MeGAG has no effect on development. Both the present work and our previous
results (Alexandre, 1978 a) indicate that a different situation is encountered in
the mouse such that selective inhibition of spermidine and spermine synthesis
by MeGAG inhibits cleavage before the occurrence of cavitation, whereas
a-MeOrn exerts visible effects on cavitation only at concentrations as high as
20mM. However, as has been discussed previously (Alexandre, 1978a), the
arrest of development cannot be ascribed to a specific inhibition of putrescine
synthesis since in this case the osmolarity of the medium had increased to
such an extent that the control embryos were also arrested in their development;
mouse embryos are known to be very sensitive to this parameter (Brinster,
1965).
On the assumption that, at least at the 2-cell stage, high amounts of putrescine
and low amounts of the two other polyamines could be present in the embryos,
these results can be tentatively interpreted as showing that putrescine can be
converted into spermidine and spermine in the presence of a-MeOrn, but not
in that of MeGAG (see Fig. 1). It seems therefore that, in contrast with the
situation described in lymphocytes stimulated by concanavalin A (Morris,
Jorstad & Seyfried, 1977), putrescine which is still synthesized in the presence
of MeGAG, can not fulfil the role played by spermidine and spermine in early
mammalian development.
It seems reasonable to believe that the cause of the arrest of development
induced by MeGAG in the present experiments is a specific decrease in
spermidine and spermine content. It has been shown that MeGAG exerts
II
EMB
53
158
H. ALEXANDRE
some pharmacological effects resulting in a decrease in RNA and protein
synthesis when it is used at high doses (millimolar level); however, when MeGAG
is used at micromolar doses, these syntheses are not affected (reviewed in
Heby et al. 1977). Our autoradiographic analysis of [3H]uridine incorporation
during cleavage inhibition by MeGAG is in good agreement with these findings
in that a decrease in RNA synthesis is only measurable when the embryos
are already arrested. In addition, a residual synthetic activity is still present
after the developmental arrest; inhibition of RNA synthesis is thus subsequent
to the induction of the arrest of the cellular cycles.
The addition of polyamines together with inhibitors of their synthesis has
often been used successfully to demonstrate the specificity of action of these
inhibitors. For instance, the effectiveness of MeGAG in the inhibition of
initiation of DNA synthesis in 3T3 cells is reduced by spermidine and spermine
(Boynton, Whitfield & Isaacs, 1976) while the inhibition of DNA synthesis in
activated lymphocytes by a combination of a-MeOrn and MeGAG is suppressed by the addition of putrescine, spermidine and spermine. A few experiments of this type have been carried out on mouse embryos; however, spermine,
as well as spermidine, are both toxic for this material at the concentrations
normally used (50-500 /m); all the treated embryos were killed and lysed
within about 10 h. Nevertheless, it has been possible to show that when the
percentage of blastocyst formation is used as the end point, spermidine at
concentrations of 10 and 20 fiu partially protects (about 50 %) against MeGAG
treatment, although spermine is far less effective (Alexandre, 1978 c).
It is interesting to find that the arrest of development corresponds to the
initiation of metabolic quiescence and not to the death of the embryos. This
was first suggested from the healthy appearance of the treated embryos and
clearly confirmed from the total absence of dead cells in the 'arrested morulae'
as shown by the absence of blastomeres stained by eosin Y and the reversibility
of the treatment up to 31 h. The transfer to fresh medium might switch on the
processes of spermidine and spermine synthesis which are required for traversing
the cell cycle in the blastomeres. However, this could be an exceptional situation,
since it has been shown that MeGAG blocks rat brain tumour cells irreversibly
in Gl and that the treatment of phytohaemagglutinin-stimulated lymphocytes
with MeGAG can only be reversed during the time where the lymphocytes
have not yet replicated their DNA (Heby et al. 1977).
Determination of blastocoele formation
The search for the signal which initiates the formation of the blastocyst
cavity has been undertaken in several ways. Experimental manipulations on
the mouse egg have shown that cavitation is not strictly dependent on the
number of blastomeres (Tarkowski & Wroblewska, 1967; Smith & McLaren,
1977). The use of cytochalasin B, which when given at the 2-cell stage, prevents
the second cleavage division without affecting DNA replication and nuclear
Polyamines synthesis and blastocyst formation
159
division, enabled Smith & McLaren (1977) to suggest a possible role for either
the number of DNA replication cycles or the nucleocytoplasmic ratio. In all
these experiments, cavitation occurred at the same time in the treated or
manipulated eggs as in the control ones, although it has been shown that
embryos cultured in vitro cavitate significantly later than embryos developed
in vivo (Smith & McLaren, 1977).
To obtain more information about the determination of blastocoele formation, it should be possible to modify the time of its appearance by the induction
of a reversible delay in cleavage. This was partially observed by Alexandre
(1974) after acute X-irradiation, but in this case, no specific effect could be
ascribed, since X-rays induce mainly irreversible damages resulting in the
death of the embryos before they cavitated (Alexandre, 19786). 5-Bromodeoxyuridine (BUdR), which has often been used for inhibiting differentiation,
both reduced the average cell number per embryo and the frequency of blastocysts (Pollard, Baran & Bachvarova, 1976). Such a treatment is also irreversible
as are many of the treatments with other metabolic inhibitors such as actinomycin D (Mintz, 1964).
In the present work, we describe a drug-induced metabolic quiescence in
preimplantation mouse embryos, which is followed by the resumption of in
vitro development until the full grown blastocyst stage. We have found that
the delay in cavitation is proportional to the duration of the treatment, and
this is precisely what one would expect if cavitation is only induced when the
embryos have undergone a sufficient number of cell divisions; from this standpoint, our results are in good agreement with Smith & McLaren's biological
clock theory. However, a significant average reduction of about 10 cells has
been found between the cell number of control and delayed nascent blastocysts.
This difference, which is identical for all of the treated embryos irrespective of
the duration of treatment from 16 to 31 h, indicates that, under those conditions
following a period of metabolic quiescence, the required number of cells can
be reduced without any apparent consequence on the cavitation process. This
reduction is however limited to about 10 cells.
Thus, while there is good evidence in favour of the importance of the
nucleocytoplasmic ratio (Witkowska, 1973; Modlinski, 1975; Smith & McLaren,
1977), the present work suggests that another factor might be the actual
trigger for blastocoele formation. Kemler et al. (1977) have beautifully shown
that since uncompacted 30-cell embryos produced by adding monovalent antibody fragments against F9 antigen (Artzt et al. 1973) are unabled to cavitate,
the compaction occurring at the 8-cell stage and involving a given cell surface
structure is an essential step in the formation of blastocysts. As MeGAG did
not interfere with the compaction of the morulae, we propose that during
metabolic quiescence, nuclear division on one hand and cytoplasmic and
nuclear maturation on the other have been desynchronized in such a way
that some cytoplasmic modifications take place in the absence of nuclear
160
H. ALEXANDRE
division. This is consistent with the early findings of Mintz (1964) who wrote
that: 'If 2-cell eggs undergo a reversible delay in development from which they
can recover within a day, and are still 2-cells the next day, RNA-synthesizing
nucleoli make their appearance, as if the normal developmental stage had
been reached'. Indeed, we have found that inhibition of the RNA synthesis
is subsequent to the arrest of nuclear divisions. In other words, an arrested
8-cell stage is morphologically more advanced than a control 8-cell stage. This
is consistent with the fact that the mean cell number of delayed nascent blastocysts is reduced at the same value for a treatment of 16 h and of 31 h.
In conclusion, the cavitation signal could be a cytoplasmic factor which is
presumably under nuclear control and which in normal conditions reaches
its maximum level at the end of the fifth cell cycle. Although this occurs at a
precise nucleocytoplasmic ratio, this ratio could not be the signal by itself.
This hypothesis might explain the fact that, occasionally, some embryos with
a very small number of cells (sometimes only two) have a vacuolated cytoplasm
and can acquire the vesicular forms described by Tarkowski & Wroblewska
(1967). This has been seen in control embryos cultured in vitro, as reported by
Tarkowski and Snow (discussion of Gardner & Rossant, 1976) and in treated
embryos similar to the few 'blastocysts' recorded in the continuously or irreversibly (41 h) MeGAG-treated embryos. It is still difficult to distinguish
between these two possibilities: either a true cytoplasmic maturation occurring
without any DNA replication or a degenerative process. Only an exhaustive
ultrastructural analysis could give an answer.
I wish to thank Professor J. Brachet and Dr Th. Vanden Driessche for helpful discussions, Dr J. Osborn for improving the English and Mr D. Franckx for help in the
preparation of the figures.
This work was supported by the European Community (Contract Euratom-ULB 099/72/
IBIAB).
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(Received 21 February 1979, revised 4 April 1979)