/ . Embryo!, exp. Morph. Vol. 34, 2, pp. 279-289, 1975
Printed in Great Britain
279
Induction of triploidy in the mouse by
cytochalasin B
By ANNA NIEMIERKO 1
From the Department of Embryology, Zoological Institute,
University of Warsaw
SUMMARY
Mouse eggs fertilized in vivo were treated with cytochalasin B in vitro (5 /tg/ml of culture
medium) at the moment of extrusion of the second polar body (2-5, 30, 3-5 h after copulation). Cytochalasin B inhibits cytokinesis of the second maturation division, so that
triploid di^vnic eggs are formed in over 50% of treated eggs.
Triploid eggs were transplanted to the oviducts of recipients. On the 4th and 5th day
of development 41-7% of transplanted eggs were recovered. All embryos recovered on the
4th day were morulae, while on the 5th day blastocysts predominated. Recovered embryos
were studied for cell number and ploidy. Twenty-three of 27 embryos with analysable
metaphase plates were triploid and four were diploid (the latter were found in females into
which both triploid and control diploid eggs were transplanted).
Sex chromosome constitution was determined in seven cases: four triploids were XXY
and three were XXX.
Preliminary observations showed that triploid embryos obtained by the use of cytochalasin B can implant and survive at least to the 9th day.
INTRODUCTION
Spontaneous suppression of the second maturation division of the oocyte is
one mechanism leading to triploidy. In an attempt to induce or increase the
frequency of this anomaly, the effect of various factors (heat-shock, colchicine,
colcemtd, pharmacological agents and delayed fertilization) on the second
maturation division of mouse, rabbit and rat oocytes has been investigated (see
Discussion).
In 1967 Carter found that cytochalasin B inhibits cytokinesis of cells without
interfering with karyokinesis. Snow (1973) and Tarkowski, Witkowska & Opas
(personal communication) induced tetraploidy in the mouse by suppressing the
second cleavage division with cytochalasin B. Inhibition of the maturation divisions in the oocytes of the snail Spisula solidissima under the influence of
cytochalasin B was described by Longo (1972). These three pieces of information suggested the use of cytochalasin B as a tool to induce triploidy in the mouse
by suppressing second polar body formation.
1
Author's address: Laboratory of Experimental Embryology, Institute of Obstetrics
and Gynaecology, 00-315 Warsaw, Karowa 2, Poland.
iS
E
M B
34
280
A. NIEMIERKO
MATERIAL AND METHODS
Spontaneously ovulating females of the inbred strain A, kept under a 16 h
light/8 h dark cycle centred on midnight, were used throughout the experiment.
Between 8.00 and 10.00 a.m. the females were paired with males and examined
at 20 min intervals for the presence of a vaginal plug (delayed matings). The
females were killed 2-5, 3-0, 3-5 h after copulation and the oocytes fertilized in
vivo were cultured under paraffin oil in drops of Whitten's medium (1971) containing 5 /Ag/ml of cytochalasin B for 5-7 h (usually 5 h). Cumulus cells were
either removed with hyaluronidase before culture or left intact. After thorough
washing the oocytes were cultured for 2 h and examined under an inverted
microscope for the presence of the second polar body and for the number of
pronuclei. Some eggs were fixed and mounted as permanent preparations; the
others were transplanted into the oviducts of Swiss albino females on the first
day of pseudopregnancy (Tarkowski, 1959). The transplanted embryos were
examined on the 4th and 5th and 9-1 lth day of development. Chromosome preparations were made of morulae and blastocysts by the method of Tarkowski
(1966).
RESULTS
When fertilized eggs are subjected to the action of cytochalasin B (CB) in
vitro at the moment of extrusion of the second polar body (2 P.B.), cytokinesis of
the second maturation division is suppressed so that triploid digynic eggs are
formed (Fig. 1).
As shown in Table 1 the yield of triploid eggs varies according to the length of
time between mating (fertilization) and treatment with CB. When oocytes were
obtained 2-5 h after copulation and treated with hyaluronidase to remove the
cumulus oophorus, only 39-0% were fertilized, but all of these were triploid.
In order to increase the number of fertilized eggs, the oocytes were subjected
to CB at 3 and 3-5 h after copulation. Although the percentage of fertilized eggs
increased to 68-1 % and 80-0% respectively, the proportion of triploids rose only
to 52-7% and 54-5%. The remaining fertilized eggs must have extruded 2 P.B.
before treatment and were diploid.
The yield of triploid embryos obtained in 3 h (cumulus present) and 3-5 h
(cumulus absent) series are similar, showing that penetration of CB into eggs is
not impaired by the presence of the cumulus oophorus. The observation of
cumulus-free oocytes in the process of extrusion of 2 P.B. showed that CB
rapidly passes through the zona pellucida and that its action is very fast. The
bulging of cytoplasm of 2 P.B. is retracted in about 2 min. When transferred
from medium with CB to normal medium the eggs react by wrinkling of the surface which lasts for 0-5-1 h. No delayed extrusion of previously suppressed
2 P.B. was observed. However, in a certain number of cases the tri-nucleate eggs
undergo fragmentation (Table 1).
Triploidy in the mouse
281
&
;
••
A
\
-If
Fig. 1. Triploid mouse egg. Long arrow: two female pronuclei. Short arrow:
male pronucleus. x 830.
Fig. 2. Triploid metaphase plate of 3-5-day-old morula. x 1230.
Fig. 3. Triploid blastocysts 4-5 days old. x480.
All triploid eggs transplanted to recipients originated from a 3 h series.
Preimplantation development of triploid embryos
Two hundred and forty-three eggs were transplanted to 32 recipients. This
number included 223 triploid eggs (91-8% of transferred eggs) and 20 diploid
eggs (8-2%). Thirteen females received both 3/i and 2n eggs and 19 received 3n
eggs only (Table 2). Twenty-ftve transplantations were successful. The recovered
embryos were subjected to colchecine or colcemid (1 /tg/ml of culture medium
for 1-2 h) and examined in air-dried preparations for the number of cells and
ploidy.
All embryos recovered on the 4th day (3-5 days old) were morulae, with not
more than 20 cells (Fig. 4). Of 24 morulae recovered on the 4th day and cultured
18-2
282
A. NIEMIERKO
Table 1. Effect of cytochalasin B on suppression of 2 P.B. and on
induction of triploidy
Time
after
mating
(h)
Cumulus
oophorus
2i
Absent
Total no.
of eggs
41
100%
3
Present
726
100%
Absent
55
100%
Unfertilized
eggs
Fragmenting
eggs
22
3*
53-6%
7-4%
211
29%
11
20%
20|
Fertilized
eggs
16
39-0%
100%
495
383
2-9%
68-1%
100%
—
—
44
80%
In eggs
16
39-0%
100%
100%
* 3/i embryos
3// eggs
52-7%
77-3%
—
112
15-4%
22-7%
30
14
54-5%
68-2%
25-5%
31-8%
t Includes eight 3/7 embryos.
in vitro for 24 h, 17 transformed into morphologically normal blastocysts
(Table 2, 2nd row).
In embryos recovered on the 5th day the 2-h culture in CB-free medium was
omitted. All but one were triploid. Twenty blastocysts and 13 morulae were
recovered. Most of the 4-5-day morulae were similar in cell number to the
3-5-day ones (Fig. 4). The 4-5-day blastocysts exhibited wide variations in cell
number (12-54); a third of them contained over 30 cells.
A total of 71 embryos were karyologically studied: 35 morulae and 36 blastocysts. Of the embryos examined, 38 had metaphase plates but ploidy could be
determined in 27 embryos only. Twenty-three embryos were triploid (Fig. 2) and
four were diploid (Table 3, Fig. 5). The sex chromosome complement of 3«
embryos was determined in seven cases: three triploids were XXX and four
XXY. The 7 chromosome was identified according to the criteria of Ford (1966).
The karyologically identified triploids and diploids constituted 11-6% and
20 % of the transplanted 3« and In eggs, respectively. On the unlikely assumption that all transplanted In eggs developed into morulae and/or blastocysts,
and hence that 16 karyologically unidentified diploids are hidden among the 33
embryos lacking metaphase plates, then the remaining 17 embryos must be
triploids. The number of recovered 3« embryos would then increase to 40,
giving a minimum estimate of 20% development of the 3/7 eggs transferred in
successful transplantations.
Karyologically identified triploid blastocysts attain on the 5th day of development a cell number similar to that found in four diploid-treated blastocysts of
the same age (Fig. 5). This suggests that the slow rate of development of triploid
13
10*
9
8*
10
7*
19
15*
32
25*
4-5, 3,5 days in vivo
+ 1 day in vitro
4-5, in vivo
Total, 4-5
Grand total
transplantations
3-5, in vivo
Age of embryos
(days) and place
of development
100%
243
199
100%
156
127
100%
87
64
100%
69
63
100%
87
72
Total
89-9%
223
179
145
116
86
63
59
53
78
63
3/7
7-9
8-6
91
7-8
7-2
Mean no.
of eggs/ $
Total
41-7%
100%
83
49-6%
100%
63
54-6%
100%
35
44-4%
100%
28
27-7%
100%
20
* Successful transplantations.
io-i%
20
20
11
11
1
1
10
10
9
9
2/7
Transplanted eggs
17
27%
19-6%
46-9%
39
15-8%
31-7%
20
20-3%
38-3%
13
*-J /o
H-1%
23°/
18-6%
44-5%
37
58-7%
37
29%
31-2%
56-1%
20
62-7%
3-5%
8-6%
7
4-8%
9-6%
6
3-1%
5-6%
2
6-3%
14-3%
4
5%
95%
7
1-3%
1
3-3
3-3
5
3-5
2
Cleaving Mean no.
of
eggs
(2-4 cells) embryos/?
26-4%
19
Morulae
Blastocysts
Recovered embryos
Table 2. Transplantation of eggs treated with cytochalasin B
OO
to
©
3
;s
*-*
©
284
A. NIEMIERKO
|
I 3:5-day morulae
4-5-day morulae
4-5-day blastocysts
10
20
30
40
50
60
Number of cells
70
80
Fig. 4. Number of cells in 3-5 and 4-5-day embryos.
embryos results from the treatment applied in vitro to the newly fertilized eggs,
rather than from the triploid condition per se.
Postimplantation development of triploid embr-yos
In a preliminary experiment, 40 tri-nucleate eggs were transplanted to six
recipients which were killed on the 9th, 10th and 11th day of pregnancy. Seven
implantations were found in two females. The implantations were opened and
inspected under a dissecting microscope. One female killed on the 9th day had
five implantations: four contained a normal egg-cylinder delayed by 1 day as
compared with normal development, and one implantation contained no
embryo. Another female killed on the 10th day had two failed implants. Although karyological studies have not been carried out on these embryos it
seems probable that they were triploid.
285
Triploidy in the mouse
3/; morulae
10
I
| 3/; blastocysts
Illl 111] 2// blastocysts
9
8 O
"*"*
7 —
E
o
"5 6 5 Z.
3 -
1 -
10
20
30 40 50
Number of cells
60
70
SO
Fig. 5. Number of cells in 3/? morulae and blastocysts and 2/7 blastocysts.
Table 3. Karyology of 3-5- and 4-5-day embryos
Number of embryos
Stage
Morulae
Blastocysts
Total
Age of
embryos Exam(days)
ined
Without
metaphase
plates
Ploidy
With
With analysable
metametaphase
phase
plates
plates
3/7
2/7
—
3-5
4-5
17
18
13
13
4
5
4
2
4
2
4-5
36
7
29
21
17
4
71
33
38
100%
46-5%
53-5%
27
38-0%
23
32-4%
4
5-6%
* Number of triploid embryos.
—
Sex
chromosome
complement
XX Y (2*)
—
XXY
XXX
XXY
XXX
(2)
(3)
(4)
(3)
286
A. NIEMIERKO
DISCUSSION
CB inhibits cytokinesis of the second maturation division of mouse oocytes
and offers an effective method of obtaining triploid mouse embryos (Table 1).
Its effectiveness in this study (over 50 % of treated eggs) is limited only by the
non-simultaneous penetration of spermatozoa into oocytes.
The first attempts to induce triploidy in mammals were performed on the
mouse by Beatty & Fischberg (1949), Fischberg & Beatty (1951 a) and Braden &
Austin (1954), who by applying heat-shock at the time of fertilization obtained
25-7% and 15-3% triploid 3-5-day-old embryos and 17% tri-nucleate eggs
respectively. Edwards (1954, 1958a, b; 1961), using colchicine and colcemid,
obtained 19% and 50% of triploid one-cell eggs, and up to 10% of triploid
embryos at 3-5 days. In similar studies McGaughey & Chang (1969) obtained
1-3-8% of triploid eggs at the first cleavage. A high incidence of triploid eggs
and embryos was recently reported by Takagi (1970) in superovulated females of
A/He strain. This is the only known case of increased frequency of triploidy
in the mouse as a result of superovulation. Piko & Bomsel-Helmreich (1960)
after colchicine treatment found 38% rat eggs with suppressed second polar
body. The highest percentage of triploid eggs - up to 97 % - was obtained by
Bomsel-Helmreich (1965) by using colcemid on rabbit oocytes fertilized in vitro,
from which 71 % developed into blastocysts.
The efficiency of previous methods of inducing triploidy in the mouse was
relatively low. Antimitotic agents like colchicine and colcemid, which have been
successfully used to induce triploidy in the rabbit, in the mouse interfere with
fertilization (McGaughey & Chang, 1969) and in a high proportion of eggs
cause scattering of the chromosomes of the second meiotic metaphase into
several groups and consequently the formation of many nuclei. Besides, these
substances, especially colchicine, are toxic because they are bound to proteins
of microtubular structures (Borisy & Taylor, 1967) and for this reason they may
interfere with further development of the eggs.
CB does not interfere with the course of karyokinesis of the second meiotic
division, so that two pronuclei are regularly formed. It also does not impair the
further development of eggs treated during cleavage (Snow, 1973; Tarkowski,
Witkowska & Opas, personal communication). In the concentration used in the
present work, CB does not interfere with the fertilization of mouse oocytes in
vitro and yields a high percentage of triploid eggs (Komar & Niemierko, unpublished observations).
Despite its high effectiveness in vitro, the attempt to suppress the formation of
2 P.B. with CB in vivo was unsuccessful. Injection of culture medium containing
5 /tg/ml of CB into the oviduct 2-5 and 3 h after mating failed to induce triploidy (Niemierko, unpublished observations).
When triploidy is induced with CB in vitro and the treated eggs are transplanted to recipients, loss of a certain proportion of eggs is inevitable. In the
Triploidy in the mouse
287
present study no eggs were recovered from seven females into which 44 triploid
eggs were transplanted. In the successful transplantations developing embryos
were found, representing 41-7% of the transplanted eggs, as well as 30 one-cell
eggs and 42 fragmenting eggs, which may have included some CB-treated eggs in
addition to native eggs of the recipients.
It seems that the slower rate of development of triploids resulted mainly from
the fact that shortly after fertilization the eggs were subjected to stress (CB,
culture in vitro, transplantation). Culture in vitro is probably responsible for the
lower cell number in 4-5-day embryos which spend the last day in culture as
compared to embryos developing 4-5 days in vivo (mean cell number 27 and 43-4
respectively). The present data do not permit the effect of triploidy itself on the
survival and rate of preimplantation development to be analysed because very
few control diploid eggs were submitted to the same treatment. However, there
are reports that triploidy may affect the survival of embryos even before
implantation. For instance, Braden (1957) observed that in 'silver' strain mice
only half the triploid eggs of various origins (suppression of 1 P.B., suppression
2 P.B., dispermy), which at the one-cell stage constituted 8.3%, reached the
blastocyst stage. This author suggests, following Fischberg & Beatty (1952),
'lower survival rate during cleavage of the inbred triploid embryos resulting
from intrastrain matings'.
The eggs used in the present study were obtained from delayed matings.
According to some authors delayed fertilization increases the incidence of
digynic and dispermic triploidy in laboratory animals and man (Schindler &
Mikano, 1970). In the mouse Vickers (1969) noted in the PDE strain a ninefold
increase of triploidy of unknown origin (2-9 % versus 0-32%). Marston & Chang
(1964) observed 4-2 % of digyny and 3-3 % of dispermy in eggs fertilized 12-13 h
after ovulation. On the contrary, no increase in incidence of triploidy was
observed by Gates & Beatty (1954) and Braden & Austin (1954). Litters of normal size were observed in spontaneously ovulating females mated in the early
morning following ovulation (Krzanowska, 1964; Tarkowski, Witkowska &
Nowicka, 1970), showing that by the time of day when the present experiments
were carried out the oocytes were fully viable and that the incidence of abnormalities of oocyte maturation and fertilization had not significantly increased.
From earlier studies it is known that, notwithstanding the way in which it
arises and the sex chromosome constitution, triploidy in laboratory animals is
lethal around the middle of gestation (rabbit, Bomsel-Helmreich, 1965; rat,
Piko & Bomsel-Helmreich, 1960; mouse, Fischberg & Beatty, 1951 b; Vickers,
1969; Wroblewska, 1971). In man triploidy is usually lethal at 6-9 weeks (Carr,
1970, 1971 #, b) but occasionally triploid foetuses can develop till birth (20
such cases have been reported). The 14 infants for which details are published
exhibited a constellation of anomalies, with no clear syndrome (Walker,
Andrews, Gregson & Gault, 1973).
Comparison of the descriptions of triploid mouse embryos given by various
288
A. NIEMIERKO
authors suggests that the morphological expression of triploidy depends on the
genetic background. Wroblewska (1971) observed a definite syndrome in
CBA/CBA-T6T6 digynic embryos, expressing itself in the arrest of development
of the embryonic part of the egg-cylinder. Morphologically normal triploid
embryos were encountered by Fischberg & Beatty (1951 b) at 9-5 days in 'silver'
strain, by Yickers (1969) at 9-5-11-5 days in PDE strain and Takagi (1970) at
7-5 days in A/He strain mice. My preliminary investigations on the postimplantation development of triploid A-strain embryos, obtained by the use of
CB, demonstrated that on the 9th day the embryos were represented by eggcylinders normally developed but delayed by one day in comparison with control
embryos. The further fate of these embryos is under investigation.
I wish to express my thanks to Professor A. K. Tarkowski for his helpful advice and
invaluable comments on the manuscript.
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{Received 29 October 1974, revised 25 January 1975)