/. Embryo/, exp. Morph. Vol. 35, l,pp. 25-39, 1976
Printed in Great Britain
25
Diploid parthenogenetic mouse embryos produced
by heat-shock and Cytochalasin B
By HANNA BAtAKIER 1 AND ANDRZEJ K. TARKOWSKI 1
From the Department of Embryology,
Institute of Zoology, University of Warsaw
SUMMARY
Swiss albino and C57BL/10 eggs from induced ovulations, and spontaneously ovulated
A eggs, were activated in vitro by a heat shock of 44 °C for 5 or 7-5 min and cultured in the
presence of 10 /tg/ml of Cytochalasin B (CB) for 5-8 h. The activation rate was about 70 %
in Swiss albino, 40 % in C57BL and 90 % in A eggs. CB suppressed second polar body
(2P.B.) formation in over 90 % of activated eggs, with the majority containing two pronuclei.
When eggs were placed in CB-free medium their surface became wrinkled and they formed
protrusions of various sizes, which in some eggs detached to form enucleate or pronucleate
cytoplasmic fragments; some eggs broke down completely into fragments. In most eggs,
however, the surface smoothed out in a few hours and suppression of 2P.B. appeared to be
permanent. The rate of development of these eggs after transplantation to the oviduct was
delayed in terms both of cell divisions and of the time of blastocyst formation. Out of 41
implants collected on the 8th—10th day of pregnancy only two healthy looking egg-cylinders
were found on the 8th and 9th day; both were retarded, at the stage characteristic for the
7th day of normal development. The reasons for delayed preimplantation development and
low implantation rate are discussed. The present experiments corroborate earlier observations that parthenogenetic mouse embryos, even if diploid, rarely survive in the uterus
beyond the egg-cylinder stage.
INTRODUCTION
During the last few years there have been several successful attempts to
induce parthenogenetic development in the mouse (reviewed by Graham, 1974;
Tarkowski, 1971, 1975). Activation in situ by electric shock (Tarkowski,
Witkowska & Nowicka, 1970; Witkowska, 1973a) or in vitro by heat shock
(Komar, 1973) or by hyaluronidase (Graham, 1970, 1971; Kaufman, 1973 c)
leads most often to the initiation of haploid parthenogenesis. In order to increase the proportion of diploid parthenogenones, Graham (1972) developed a
technique of subjecting cumulus-free eggs to hypotonic shock, which gives a
high yield of potentially diploid eggs due to suppression of the second polar
body. This technique has been also used with success by other authors
(Graham & Deussen, 1974; Kaufman & Gardner, 1974; Kaufman & Surani,
1974). Another attempt to produce diploid parthenogenones is presented in
1
Authors' address: Department of Embryology, Institute of Zoology, University of
Warsaw, 00-927/1 Warszawa, Poland.
26
H. BALAKIER AND A. K. TARKOWSKI
this paper. We have taken advantage of the observations that Cytochalasin B
inhibits second meiotic division of the mouse oocyte (Niemierko, 1975) and
cleavage divisions (Snow, 1973; Tarkowski, Witkowska & Opas, in preparation) and that short treatment of eggs with this drug does not impair further
development. Diploid parthenogenetic embryos obtained by suppression of
second polar body with Cytochalasin B can develop beyond implantation, but
so far we have not observed survival beyond the early egg-cylinder stage.
MATERIAL AND METHODS
Eggs were obtained from Swiss albino females (randomly bred colony) and
C57BL/1O and A females. With the exception of A females which ovulated
spontaneously and mated with vasectomized males, all others were induced to
ovulate (5 i.u. of PMSG and HCG given at an interval of 45-48 h) and were
kept apart from males.
Eggs were activated in vitro within the excised oviducts by a heat shock of
44-0 ± 0-1 °C (Komar, 1973). Eggs from induced ovulations were shocked
12-20 h post-HCG and those from spontaneous ovulations between 8.00 and
9.00 a.m. Some eggs of A origin were heated for 7-5 min; all other eggs received
a shock of 5 min duration.
After the shock eggs with the cumulus oophorus intact were placed in tubes
with 0-5-1 ml of Whitten's medium (Whitten, 1971) containing 10//g of Cytochalasin B (CB)/ml. This concentration was worked out in our earlier experiments on suppression of cleavage divisions (Tarkowski et af., in preparation)
and was found effective also by Snow (1973). The tubes were gassed with 5 %
CO2 in air and kept at 37 °C. Observations of Niemierko (1975) and other
pilot experiments carried out in this laboratory showed that when newly fertilized or activated eggs are kept in CB for less than 4 h many of them subsequently
undergo fragmentation. Heat-activated eggs were therefore subjected to CB
for 5-8 h. Subsequently cumulus was removed with hyaluronidase and the
eggs placed in drops of CB-free Whitten's medium under liquid paraffin for
\-A h. During this period the eggs were examined under the inverted microscope for signs of activation and type of reaction. Some of these eggs were
subsequently fixed and mounted as permanent preparations (Tarkowski &
Wroblewska, 1967).
The activated eggs with second polar body (2P.B.) suppressed were transplanted to the oviducts of Swiss albino recipients on the first day of pseudopregnancy (day of vaginal plug = first day) or to sexually immature females
(4-5 weeks old) using the technique of Tarkowski (1959). For transplantation
we used only eggs which were collected 14-16-5 h post-HCG (in the majority
of experiments, 15-16 h).
From a number of recipients the transplanted parthenogenetic eggs were
recovered after 72-96 h (4th and 5th day) and cultured for another day in
Diploidparthenogenetic mouse embryos
27
Whitten's medium under liquid paraffin. Colcemid (1 /^g/ml) was added to
culture drops 2-3 h before making air-dried preparations (Tarkowski, 1966).
In order to evaluate whether or not the rate of preimplantation development
of parthenogenones is delayed, the stage of development and the number of
cells were estimated in F1 (Swiss ? x A ( J ) embryos obtained on the 4th day after
night matings and after delayed matings (7.00-9.00 a.m.), i.e. at the time at
which activation was performed.
Another group of recipients (only mature pseudopregnant females) were
killed on the 8th—10th day and inspected for signs of implantation. Most implantation swellings recovered on the 9th day and all from the 10th day were
torn open and inspected under the dissecting microscope. Eight-day and some
of the 9-day implantations were examined in routine haematoxylin and eosin
histological preparations.
As control experiments, eggs harvested 12, 16 and 20 h post-HCG were
subjected to heat shock and cultured in CB-free medium, and non-heat-treated
eggs were cultured in medium containing CB. The control eggs were fixed after
the same period of time as the experimental ones and mounted in permanent
preparations for examination.
RESULTS
Control experiments
Reaction of non-heated eggs to culture in CB
As can be seen in Tables 1 and 2, over 90% of eggs cultured in the intact
cumulus oophorus remained in metaphase II. The incidence of activated eggs
did not exceed 3 % , but a number of eggs underwent complete fragmentation
or were degenerate. Thus culture in CB has no appreciable effect per se on
activation.
Reaction of eggs to heat shock alone {culture in CB-free medium)
In order to evaluate properly the effect of CB on activated eggs, over 800
Swiss albino eggs were subjected to heat shock alone and fixed for examination
as permanent preparations 4-9 h later (Table 1). With the exception of eggs
treated 12 h post-HCG, the overall activation rate in 15-16 and 20-h-old eggs
was about 70%, which corresponded well to the activation rate in the experimental series (cf. Tables 1 and 2). Because of the low activation rate in the
12-h group, eggs used in experiments with CB were 13-16-5 h old. The present
observations confirmed the fact, noted already by other authors, that with
advancing age the eggs become more susceptible to activation but at the same
time the incidence of 'fragmentation' increases. As already shown by Komar
(1973), eggs subjected to heat shock react most often by extrusion of 2P.B. and
formation of a haploid pronucleus and by immediate cleavage. Because
immediate cleavage is sometimes irregular (the two cells are of uneven size
3-8
6-4
13-16
20
320
15 3
t PR = pronucleus.
6-3
23-3
6-3
14-2
236
12-6
10
230
16
6-6
9-9
14-2
191
(Series II) X
15-16 494
(total)
9-6
8-6
303
3-5
15-16
(Series 1)J
640
ing
mpn f_
JHCill
Frag-
114
0
generated
vated
clL-ll
11U. Ul
eggs
De-
Non-
Total
12
(h)
* 2P.B. = second polar body.
Heat-shock, 5-8 h culture
in CB, 0-4 h culture in
CB-free medium
Heat-shock, 4—9 h culture
in CB-free medium
Treatment
postHPCr
Age
660
670
1000
8-3
0
4-8
3-2
5-9
2-7
0-9
47-8
43-8
571
35-6
9-6
7-6
7-9
7-9
7-8
10-8
0-9
29-3
33-2
27-8
36-6
32-4
0-4
8-9
0-3
0-5
163
—
156
—
156
—
31
—
2-6
—
2-6
—
Artivated
K
96-3
—
96-8
—
96-8
—
vated
Non-
Control
Frag-
0-6
—
0-6
—
0-6
—
ing
11 I d 1 L
t Series I and II were performed in identical manner, but several months apart.
800
6-4
100
40
13-7
0
32-5
1000
67-7
1000
2PRf
54-1
absent,
many Immef
2P.B. nuclei diate Enuc- Total
oo „
i
(sub- L-lCciv- lJCcl*
1 PR 1 PR nuclei) age
eggs
tion
or
2P.B.
present
Activated eggs
2P.B.*
suppressed
Total
1000
66-5
1000
71-9
1000
Experiment
Table 1. Effect of heat-shock with or without CB on Swiss albino eggs, estimated on fixed and stained eggs (%)
^\
&
H
•
0
>
>
h i
w
;>
to
oo
Swiss
albino
C57BL/10
Strain
106
208
16-16-5
Total
102
1031
14-16-5
15
Total
no of
eggs
Age
postHCG
(h)
10
—
2-8
1-4
72-5
29-2
50-5
6-3
11-3
3-8
3-3
18-2
Nonacti- Degener- Fragvated
ated menting
Total
77-0
850
59-3
89-6
2 PRf
161
8-3
33-3
5-2
1 PR
4-6
50
3-7
4-2
No. of
PR unknown
2P.B.* suppressed
Activated eggs
* 2P.B. = second polar body.
t PR= pronucleus.
74-7
1000
26-5
100-0
56-6
1000
41-8
1000
Experiment
0-3
2P.B.
+
1 PR
2-3
1-7
3-7
0-6
34
460
Immed- r
iate
Total
cleavno of.
age
eggs
—
2-8
Activated
73-5
93-7
Nonactivated
Control
Table 2. Effect of heat-shock and Cytochalasin B on Swiss albino and C57BL/10 eggs, estimated on eggs
in the living state 1-4 h after the shock (values in %)
26-5
3-5
Fragmenting or degenerated
S:
30
H. BALAKIER AND A. K. TARKOWSKI
and may be accompanied by enucleate cytoplasmic fragments) classification of
such eggs either as undergoing immediate cleavage or fragmentation becomes
somewhat arbitrary.
In Table 1 we included separately the results of two identical series of experiments carried out several months apart, in order to show that even when all
the parameters under control remain constant and the number of eggs investigated is reasonably high (200 and 300) there may be significant differences
in the proportions of eggs entering various routes of development. For instance,
the incidence of suppression of 2P.B. varied between 19-6% and 7-2% and of
extrusion of 2P.B. between 35-6% and 57-1 %.
Contrary to the similar experiments by Komar (1973), subnuclei were formed
in less than 10 % of eggs. We are unable to explain this difference.
Experiments with CB
Immediate reaction of eggs to heat shock and CB
Eggs subjected to this double treatment were examined in two ways. First,
320 Swiss albino eggs after being transferred for 0-4 h to CB-free medium
were fixed and examined as permanent preparations (Table 1). Second, 1031
Swiss and 208 C67BL/10 eggs were transferred for 1-4 h to CB-free medium
and were examined in the living state only (Table 2). After examination some
eggs from this group were selected for transplantation (see below). From both
series of experiments it is evident that CB effectively suppresses formation of
the 2P.B. (over 90%), the most common reaction being formation of two
pronuclei. As mentioned above, suppression of 2P.B. can occur after heat
shock alone, but the frequency of this phenomenon never exceeds 20 %.
In C57BL/10 eggs examined in the living state only, the activation rate among
eggs shocked 16-16-5 h post-HCG was twice as high as among eggs treated
at 15 h. The significance of this difference is uncertain. As in Swiss albino eggs,
in the majority of C57BL/10 eggs, 2P.B. was suppressed by CB.
Spontaneously ovulated eggs of A origin subjected to 5 min heat shock
between 8.00 and 9.00 a.m. reacted only in 10% (4 out of 40). However, over
90% were activated when the duration of the shock was prolonged to 7-5 min
(60 out of 65). Data concerning activation of A eggs are not included in Tables
1 and 2.
As long as the activated eggs stay in CB their surface remains completely
smooth. However, when placed in CB-free medium the surface becomes
wrinkled and forms protrusions of various size (Figs. 1, 2) which may - though
not necessarily - eventually detach to form small cytoplasmic globules lying
freely in the perivitelline space. Sometimes an indentation is formed in the
presumptive region of the second polar body formation so that the egg acquires
a bean-like shape (Fig. 1). In such a case the pronuclei may be lying on the top
of the two protrusions which are formed on both sides of the indentation
Diploid parthenogenetic mouse embryos
Fig. 1. Swiss albino eggs kept in CB for 5 h 10 min and subsequently in CB-free
medium for 3 h 20 min. The surface of eggs is still wrinkled. The upper right egg
shows an indentation in the presumptive region of the second polar body formation; the two protrusions are occupied by pronuclei. The egg shown in the middle
has extruded several small enucleate fragments and a large one with a pronucleus.
x 200.
Figs. 2, 3. Swiss albino eggs kept in CB for 6 h .10 min and in CB-free medium
for 30 min.
Fig. 2 Five activated eggs and one non-activated (top middle). The surface of the
activated eggs is nearly smooth and the eggs are only slightly deformed. In the
middle egg of the lower row the two pronuclei have already moved to the centre.
Note that the non-activated egg is round and has a completely smooth surface,
x 200.
Fig. 3. Another group of eggs from the same experiment. The surface of eggs is
smooth but the two pronuclei still remain close to the egg surface. The egg on the
right has retained the first polar body, x 400.
31
32
H. BALAKIER AND A. K. TARKOWSKI
(Fig. 1). If the furrow is not formed the two pronuclei very often lie side by
side and stay close to the egg surface (Fig. 3). Similar surface reactions are
observed when cleaving mouse eggs are subjected to CB at the time of cleavage
and subsequently placed in CB-free medium (Snow, 1973; Tarkowski et ai,
in preparation).
With time the surface of activated eggs steadily smooths out (Fig. 2). However, a number of eggs undergo severe fragmentation or extrude cytoplasmic
fragments which may even contain pronuclei (Fig. 1). This explains a small
number of eggs with 2P.B. or undergoing immediate cleavage which are
recorded in Tables 1 and 2. These phenomena occur only after the eggs are
removed from CB.
The behaviour of eggs appeared to depend on the duration of treatment with
CB. After 5-h treatment the egg surface was very wrinkled and they required
a few hours to acquire normal appearance; the incidence of fragmentation was
highest in this time group. After 8-h treatment the surface was smooth or
nearly smooth and the eggs needed a much shorter time to regain their normal
appearance. For practical reasons treatment of 6-h duration was usually
applied. Whether the longer times of exposure of eggs to CB were more harmful
for their survival could not be determined in the present study. To make sure
that the eggs with 2P.B. suppressed would remain in this state after transplantation, the eggs were kept in CB-free medium for as long as was feasible (the idea
being to complete the experiment in one day and thus to reduce the length of
culture to a minimum) and only those which looked normal, i.e. which did not
extrude cytoplasmic fragments and clearly contained one or two pronuclei,
were selected for transfer.
Contrary to the activated eggs, the surface of the non-activated eggs remained
all the time completely smooth. The different behaviour of these two types of
eggs permits them to be easily segregated even if the pronuclei are not yet
clearly visible (Fig. 2).
Preimplantation development of transplanted eggs
Healthy-looking Swiss albino eggs with 2P.B. suppressed and with two
pronuclei (occasionally with one pronucleus) were transplanted to pseudopregnant or sexually immature recipients and recovered on the 4th or 5th day.
The embryos were subsequently cultured for 24 h. On the 4th day the most
advanced embryos were morulae (Fig. 4). Blastocysts were first observed on
the 5th day; some of them transformed from morulae after 24 h in vitro (Fig. 5).
All morulae and blastocysts containing analysable metaphase plates (15 out of
27 studied) were diploid.
The tempo of development of parthenogenetic embryos was clearly retarded:
formation of blastocysts was delayed until the 5th day and the mean cell number
of 4-5- and 5-5-day-old parthenogenones corresponded to that of 3-5-day-old
controls from delayed matings (Table 3). On the 4th day the mean cell number
33
Diploid parthenogenetic mouse embryos
Figs. 4, 5. The same group of seven Swiss albino parthenogenetic embryos after
recovery from the recipient on the 4th day (Fig. 4) and after 24 h in culture
(Fig. 5). During the culture phase one egg remained at the 8-cell stage and two
morulae transformed into blastocysts. x 200.
Fig. 6. An egg-cylinder recovered on the 8th day following transfer of activated
eggs of A strain. The section is nearly perpendicular to the long axis of the cylinder.
Pro-amniotic cavity is clearly visible, x 200.
Fig. 7. A C57BL/10 egg-cylinder recovered on the 9th day. The section is again
nearly perpendicular. Although the embryo is much larger than that shown in Fig. 6,
it represents a similar stage of development. By the time of examination Reichert's
membrane must have undergone disruption, x 200.
and the proportion of embryos that had reached the blastocyst stage was considerably lower in embryos originating from females mated in the morning
following ovulation than from females mated at night (Table 3). It follows from
this that the proper control material for parthenogenetic embryos are the
embryos developed from eggs fertilized as a result of delayed matings which
take place at the time when artificial activation is carried out. However, even
if this correction is taken into account the parthenogenones still fall behind
normal embryos.
3
EMB
35
Control embryos
?$ mated at
night with Ac?c?
Control embryos
$$ mated with
AcJc? 7.00- 9.00
a.m. (delayed
matings)
Parthenogenetic
embryos
Material
18
28-1 %
16
33-3%
64
5
5
4th
5th
48
42
4th
—
6
4th
7
Total
47
c
—
Day of No. of
autopsy females
No. of
transferred
eggs
i. ^ KJ• d l l
l / \ J V t l \~>\£
4
0
18
12
15
35-7%
33
70-2%
B
27
64-3%
14
29-8%
M
A
morulae (M°) and blastocysts (B)
7
9
8
—
—
10
—
B
—
M
A
after 24 h
culture in vitro
6th
5th
4th
4th
16-9
±2-2
210
±3-1
23-3
±1-5
24-8
±2-1
Day of
develop- <Mean
±S.E.
ment
A
M
14-29
8-29
13-45
15-47
Range
29-4
±3-0
31-8
±6-2
30-2
±0-4
47-2
±2-3
+ S.E.
Mean
A
B
20-53
15-41
27-32
30-78
Range
±5-5
22-4
±2-4
27-0
25-7
±11
40-5
±2-3
+ S.E.
Mean
A
14-53
8-41
13-45
15-78
Range
Overall
Cell number in M and B at the end of experiment
Table 3. Preimplantation development of Swiss albino control fertilized eggs and of transplanted diploid parthenogenic eggs
X
o
t*
H
d
>
w
n
>—1
w
70
48
226
19
No. of
transfers*
No. of horns
with implants
23
32-9%
2
4-2%
16
7-1 °/
Total
34-8%
14-3%
16-7%
In successful
transfers
3
2
3
No. of
embryos f
12x8
2x9
5x10
2x8
1x9
1x10
2x8
3x9
1x10
No. of females
autopsied
on a given day
* Eggs were transferred either into one or into both oviducts.
t All embryos were retarded in development and only two looked healthy (one A embryo recovered on the 8th day and one
C57BL/1O recovered on the 9th day).
C57BL/1O
Swiss albino
Donor strain
No. of
transferred eggs
No. and % of implantations
Table 4. Implantation of parthenogenetic embryos in Swiss albino pseudopregnant recipients
1
I
3
36
H. BALAKIER AND A. K. TARKOWSKI
Development after implantation
Altogether 41 implantation swellings were available for examination (Table
4). The implantation rate was generally very low but clearly depended on the
donor strain. The highest rate was observed after transplantation of C57BL/10
eggs (32-9%); Swiss albino eggs and A eggs implanted only in 7-1% and
4-2% respectively.
However, although C57BL/10 eggs were superior to Swiss and A eggs as far
as the implantation rate was concerned, their subsequent survival was equally
poor (Table 4). Embryos could not be found in any swellings collected on the
9th and 10th day and inspected under the dissecting microscope. In histologically
analysed implants eight embryos were found - five on the 8th day and three on
the 9th day. Only two embryos - one A and one C57BL/1O from the 8th and
9th day respectively - looked normal and healthy. However, even these two
embryos were retarded, as they were in the stage of an egg-cylinder typical for
the 7th day of normal development (Figs. 6, 7). The remaining embryos were
clearly abnormal and checked in development at the stage of an early 6-day
cylinder.
DISCUSSION
Heat shock of 44-0 °C followed by short-term culture in the presence of
10 ^g/ml of Cytochalasin B appears to be another effective method of producing
diploid parthenogenetic mouse embryos (cf. Graham, 1972; Graham & Deussen,
1974; Kaufman & Gardner, 1974; Kaufman & Surani, 1974). CB suppresses
second polar body formation in nearly 100% of activated eggs. Depending on
the strain used (Swiss albino or C57BL/10) and the way of examination (fixed
and stained eggs or living eggs) the percentage of eggs with two pronuclei
varied between 80 and 90, and of those with one diploid pronucleus between
5 and 16. The incidence of eggs containing small subnuclei as well as one or
two pronuclei was estimated to be about 10%. Since the same frequencies were
observed in eggs cultured in CB-free medium, formation of subnuclei must be
a result of heat shock rather than CB. These eggs are potentially hypodiploid.
We have also observed that in many eggs the two pronuclei were of different
size, which suggested that at the 2nd meiotic division the chromatids were
unequally distributed. This phenomenon was observed with the help of chromosomal studies of the first cleavage of eggs activated in vitro with hyaluronidase
and hypotonic shock (Graham & Deussen, 1974). However, when 2P.B. is
suppressed, as was the case in the present experiments, aneuploidy of pronuclei
has probably no further implications, as diploidy will be restored at the first
cleavage. The fact that all morulae and blastocysts containing analysable
metaphase plates were diploid proves that suppression of 2P.B. due to CB was
permanent and confirms earlier observations that both pronuclei contribute
chromosomes to one metaphase plate of first cleavage (Kaufman, 1973&;
Diploid parthenogenetic mouse embryos
37
Witkowska, 1973# and personal communication; Graham & Deussen, 1974).
We believe, therefore, that in experiments aimed at inducing diploid parthenogenesis, aneuploidy - strictly speaking hypodiploidy - is much more likely to
arise when subnuclei are formed than when there is uneven distribution of
chromatids between the two pronuclei.
The tempo of preimplantation development of transplanted eggs (number of
cleavages and the time of transformation of morulae into blastocysts) was in
the present study rather low and unsatisfactory. There are four factors which
might have been responsible for this state of affairs. First, damage produced to
eggs by heat shock; second, harmful effect of treating the eggs with CB; third,
shock imposed on eggs at the one-cell stage by handling and culturing them
for several hours in vitro; and fourth - probably of minor importance - the
effect of second handling and culturing for the last 24 h of the experiment.
Although all these four factors might have been important, we believe that the
second and the third factors were of major importance. Support for this view
comes from the observations by Niemierko (1975) that when newly fertilized
mouse eggs are subjected to CB in vitro for 5-6 h before being transplanted to
the oviduct, the survival of embryos is affected and the rate of development is
clearly delayed so that blastocysts do not form until the 5th day.
The low rate of implantation of the Swiss albino and A eggs was probably
due to different causes. The A eggs were heated for 7-5 rather than 5 min,
which might have had an adverse effect on their survival. As far as Swiss albino
eggs are concerned their preimplantation development is generally slow, and
these embryos were handicapped when transferred to the synchronized recipients
of their own strain. In other words, in the combination of transfer 1 -» 1, the
parthenogenetic embryos might not have been able to 'catch up' with the
uterus, the more so since their preimplantation development was retarded.
Unlike eggs of many other strains including Swiss albino, fertilized C57BL/10
eggs develop fast (cf. table 1 in McLaren & Bowman, 1973 and Table 3 in the
present paper) and probably for this reason the parthenogenetic ones were
more successful in implantation. Additional evidence for this supposition comes
from the observation that the decidual swellings induced by C57BL/10 parthenogenetic embryos were regularly larger than those induced by Swiss albino
and A embryos.
The inability of diploid parthenogenones to survive in the uterus beyond the
egg-cylinder stage remains a mystery. Our present results are concordant with
earlier observations on post-implantation development of experimental parthenogenones presented by Tarkowski et al. (1970), Graham (1972), Witkowska
(19736), Mintz & Gearhart (1973) and Kaufman & Gardner (1974) as well as
of spontaneous parthenogenones (Stevens & Varnum, 1974). The most relevant
to the problem under consideration are the papers by Graham and by Kaufman
& Gardner, because these authors dealt exclusively with diploid eggs rather
than a mixed population of haploids, diploids and n/2n mosaics, so the mortality
38
H. BALAKIER AND A. K. TARKOWSKI
of embryos which they observed could not be due to haploidy. As suggested
already in 1970 by Tarkowski et ah, diploidy per se is not a sufficient condition
for the survival of parthenogenones beyond the egg-cylinder stage. At present
there is no satisfactory explanation for the death of these embryos at this
particular stage (for a more detailed discussion see Graham, 1974; Tarkowski,
1975). The possibility that embryonic death is due to lethal genetic factors
which at this stage start to operate at the cellular level can be rejected because
both experimentally produced and spontaneously arising mouse parthenogenones when transplanted to ectopic sites can grow and give rise to various
differentiated tissues (Graham, 1970; Stevens & Varnum, 1974; lies et ah 1975).
Also, parthenogenetic origin does not affect trophoblast, in that giant cells are
present in embryos growing in the uterus as well as in ectopic 'growths'.
We wish to thank Mrs A. Szarska for skilful technical assistance and Mr P. Halkiewicz
for preparing photographic documentation.
This work was partly financed by the Zoological Committee of the Section of Biological
Sciences of the Polish Academy of Sciences.
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{Received 16 April 1975, revised 2 July 1975)