J. Embryol exp. Morph. Vol. 66, pp. 81-89, 1981
Printed in Great Britain © Company of Biologists Limited 1981
Postimplantation development of CB-induced
triploid mouse embryos
By ANNA NIEMIERKO 1
From the Laboratory of Experimental Embryology, Institute of
Obstetrics and Gynaecology, Medical Academy, Warsaw
SUMMARY
By subjecting A strain eggs at the time of fertilization and polar body extrusion to 5 fig/ml
cytochalasin B, digynic triploidy was produced in 80 % fertilized eggs. Triplonucleate eggs
were transplanted to recipients and examined between 9-1 lth day of pregnancy. Development of triploid mouse embryos up to day 7 is normal and most embryos form early egg
cylinder. At day 8 the embryonic part of the cylinders is under-developed and later development fails to form an embryo. Development of foetal membranes is much less affected,
CB-induced triploids survive to 10th- day of pregnancy.
INTRODUCTION
Mammalian embryos of abnormal ploidy have been used in developmental
genetics research. This approach involves genome manipulation and experiments on effect of various types of heteroploidy on embryonic development.
Efficient methods of obtaining mono- and trisomic mouse embryos (Gropp,
1975, 1976, 1978; White, Tijo, Van der Water & Crandall, 1914a, b), gynogenetic hyphohaploid, haploid and diploid embryos (Kaufman & Sachs, 1975;
and literature review: Graham, 1974; Kaufman, 1975, 1978; Tarkowski, 1975;
BaTakier & Tarkowski, 1976) and haploid embryos from fertilized eggs
(Modliriski, 1975; Tarkowski, 1977) have been worked out.
Cytochalasin B turned out to be a useful tool in producing polyploidy in the
mouse; it proved to be superior to all the other methods employed in production
of triploidy (Niemierko, 1975; Niemierko & Komar, 1977) and tetraploid
embryos (Snow, 1973; 1975; 1976; Tarkowski, Witkowska & Opas, 1977). The
other methods for inducing triploidy and tetraploidy in mice are described by
Niemierko & Opas (1978).
The attempts to induce triploidy in mammals were carried out in rat, rabbit
and mouse (see Discussion). Most of experiments on triploidy in mouse dealt
with preimplantation period (for references see Niemierko, 1975). The viability
1
Author's present address: Department of Histology, Institute of Biostructure, Medical
School, Chalubiriskiego 5, 02-004 Warsaw, Poland.
82
A. NIEMIERKO
Table 1. Postimplantation development of CB-induced triploid mouse
embryos
Ploidyofeggs
Total 3n
Control
11
12
—
—
—
65
20
—
10
92
75
3n
Age of embryos (day)
No. of eggs
No. of implantation (%)
No. of implantation
without embryo
with embryo
Embryos examined
histologically
karyologically
Ploidy of embryos
9
13
5
(38,5)
10
40
15
(37,8)
(81,0)
1
4
5
10
—
—
6
14
2
73
—
4
4
6
—
—
—
4
10
5
67*
6
4xn.d
5x 3n
1 xn.d.
n.d.: not detected, not analysable metaphase plates.
* 65 embryos examined morphologically.
6x2n
of embryos carrying this aberration was only estimated in postimplantation
period (see Discussion). Morphological expression of spontaneous triploidy in
mouse was rarely analysed in detail (Wroblewska, 1971; Baranov, 1976). The
present study describes postimplantation development of cytochalasin Binduced triploid embryos in the mouse.
MATERIALS AND METHODS
Triploid dygynic mouse oocytes were obtained by inhibiting cytokinesis of
the 2nd maturation division in fertilized oocytes by means of cytochalasin
B (Niemierko, 1975). Oocytes of A strain from spontaneous ovulation, fertilized
in delayed, controlled matings, were used. Three hours after the vaginal plug
was found, eggs were released from oviduct, cleaned of follicular cells and put
Fig. 1 and'4. 10-day-old control embryos of normal structure and size, a-head
fold/s/, b-fore gut, c-hind gut, d-heart, e-amnion, f-somites.
Fig. 2, 3. 5. Section through the 10-day-old triploid embryos developed from
tripronucleate aggs obtained by CB.
Fig. 2. Triploid embryo No. 1 on the stage of amnion formation, a-ectoplacental
cavity, b-extraembryonic cavity, c-amniotic cavity, d-retarded and underdeveloped
embryonic part.
Fig. 3. Triploid embryo No. 2. 10-day-old triploid yolk sac without embryo.
Fig. 5. Triploid egg cylinder No. 3. with normally formed foetal membranes and
rudimentary region of the embryo, a-amnion, 5-allantois, c-head folds, d-foregut
pocket.
Fig. 6. Magnification of the head folds of embryo No. 3. Arrows-mitosis in neural
ectoderm.
Triploidy in the mouse
^•>^^-J,3*
10 mm
83
84
Triploidy in the mouse
85
in drops of medium with cytochalasin B (5 /*g/ml) under liquid paraffin. After
culture eggs were examined under the inverted microscope for the presence of
three pronuclei and triploid ones were transplanted to recipients in first day of
pseudopregnancy. Control experiments involved transplantation of diploid eggs
in which cytochalasin B did not block the 2nd polar body extrusion. Postimplantation development was examined on 9th, 10th, and 11th day. Implanted triploids and some diploids embryos were fixed in Bouin fixative, cut
in 6 fim section and stained with haematoxylin and eosin or, after being dissected from uterus, karyologically. Chromosomal preparations from some
triploid and control egg cylinders were made using the technique of Evans,
Burtenshaw & Ford (1972). Remaining control embryos were studied morphologically. Their development was assessed by comparison of morphology with
the embryos of equivalent post-coital age and on the basis of data given by
Snell & Stevens (1966), and Theiler (1972).
RESULTS
Sixty-five triploid and 92 diploid eggs were transferred to oviducts (Table 1),
both kinds of eggs being transferred separately to different recipient females.
After transplanting triploid eggs five implantations, one lacking an embryo,
were found on the 9th day of development. They contained four embryos at an
early egg-cylinder stage. The embryos looked normal although they were
smaller than controls of the same age. Their ploidy was not assessed, but they
can be classified as triploids, because block of 2nd polar body is irreversible
(Niemierko, 1975). Moreover, selection of eggs for transplantation was very
rigorous.
Fifteen implantations were found on the 10th day of development, but only
10 of them contained egg cylinders. Among six embryos studied karyologically
in all but one case mitotic plates with 60 chromosomes were found. The genetic
sex of those embryos was not determined.
Four egg cylinders examined histologically were different from each other.
One of them was an empty yolk sac with no embryo inside (Fig. 3). The remaining three egg cylinders contained embryos in a pre-somite stage (Fig. 2, 5, 7-11).
Egg cylinder No. 1. (Fig. 2) was at the stage ofamnion formation with a distinctly
shortened embryonic part. The embryo had a shortened ectoplacental cone
in form of loose group of cells. Its stage corresponded to the early 8-day A strain
diploid embryo. Egg cylinder No. 3 (Fig. 5, 6) was the most advanced in
Fig. 7-11. Triploid embryo No. 4 with rudimentary embryo.
Fig. 7. Long arrow-remnant of uterine lumen, short arrow-some scattered cells of
ectoplacental cone, a-chorion, b-amnion.
Fig. 8-10. a-amnion, b-rudimentary allantois, c-neural plate.
Fig. 11. Higher magnification neural plate of embryo No. 4 a-neural plate,
b-rudimentary mesoderm of embryo.
86
A. NIEMIERKO
organogenesis found in present study; it contained an incomplete embryo.
Only the head part of this embryo, in form of headfold primordia, was developed.
Also the foregut pocket could be clearly seen (Fig. 5). The embryo had regular
membranes: two-layered amnion and allantois separated from ectoplacental
cone and contained rudimental heart mesoderm. Dividing cells were visible in
neural ectoderm and allantois (Fig. 6). Egg cylinder No. 4. contained no
developed embryo. Scanty embryonic ectoderm in a neural plate stage (Fig. 7-11)
and scanty mesoderm adhering to it could be seen. The cylinder contained twolayered amnion, rudimentary allantois and poorly developed ectoplacental
cone (Fig. 7).
After transplanting control CB-treated eggs 75 implantation (85 % of transplanted eggs) were found on day 10 of development. Diploid cylinders were
retarded by 0-5-1 day in their development. An embryo was lacking only in two
cases and in one case implantation in form of trophoblastic giant cells was
found (2 % of transplanted eggs). Each of six karyologically studied control
embryos was diploid. Embryos studied histologically were normal: they were
either in process of turning (Fig. 1) or prior to turning (Fig. 4).
On day 11 of development no implantation or resorption after transplanting triploid eggs was obtained.
DISCUSSION
Triploid embryos produced by means of cytochalasin B are capable of
implanting. The yield of triploid implantations found in this study (37 %) is
similar to the yield of triploid blastocysts developed from CB-produced triploids
eggs (Niemierko, 1975). This allows one to suppose that irrespective of the further
course of development, all triploid 5-day embryos have the capacity to implant.
Up to date studies on triploidy in mouse indicate that this aberration may
express itself in various ways show a whole range of anomalies: from egg
cylinders as vesicles composed of foetal membranes, to a very few with embryonic structures at different stages of development.
Anomalies of triploid embryos become distinct only after 8th-9th day of
development. The early triploid embryos, i.e. 6-5-7-5 days old, showed slight
retardation, with no manifestation of anomalies in organogenesis of ectodermal
and endodermal structures and therefore they were classified as normal
(Fischberg & Beatty, 1951; Takagi, 1970; Takagi & Oshimura, 1973; Takagi &
Sasaki, 1976; Baranov, 1976). Also, in the present study 9-day triploid embryos
were morphologically normal although smaller.
Histologically studied, 10-day-old A strain triploid egg cylinders are comparable in size to early 8-day diploid egg cylinders of this strain. Triploid egg
cylinders display characteristic developmental anomalies, comprising total lack
or poor development of embryonic structures, with mesodermal derivatives
being affected to a large extent. Foetal membranes of those egg cylinders are
Triploidy in the mouse
87
developed, although in size and proportion they seldom correspond with the
embryos age. In none of the embryos examined was the allantois fused with
ectoplacental cone. A properly developed ectoplacental cone was found only
in one case (Fig. 3).
Similarly affected is the development of triploids of CBA strain (Wroblewska,
1971) and tetraploid CB-induced tetraploids described by Tarkowski et al.
(1977).
Postimplantation mortality of triploid embryos in the mouse occurs in two
specific periods. The majority of triploid ceases to develop around the 8th day
and soon degenerate. In those few strains where triploids achieve relatively
advanced organogenesis, they do not survive after the 12th day of pregnancy
(Vickers, 1969; Takagi & Oshimura, 1973; Wroblewska, 1971, 1978; Baranov,
1976; Opas, 1977). Triploid embryos of other laboratory animals also survive
only until mid-pregnancy. Triploid embryos of the rabbit survive until the 17th
day (Bomsel-Helmreich, 1965, 1971), those of rat-until the 12 day (Piko &
Bomsel-Helmreich, 1960).
The reasons for abnormal development of triploid mouse embryos remain
still unsolved, and only for triploids with XYY might it be attributed to sex
chromosome set (Walker, Andrews, Gregson & Gault, 1973 Fryns, Van der
Kerchhove, Goddeens & Van de Barghe, 1976; Kajii & Nikawa, 1977; Beatty,
1978).
Disorganized growth and limited life span of triploids reflects abnormalities
in their cell properties, which affect cellular proliferation, differentiation, and
cellular interactions.
Studies by Wroblewska (1978) suggest the influence of genotype on development of mouse triploids. Among spontaneous triploids derived from 16 genetic
combinations, the most advanced organogenesis and highest viability was
achieved by triploids of 129/Sv strain and its crosses. Morphologically variable
expression of triploidy in different mouse strains (CBA, A-cylinders with no
embryos; 129/Sv and its crosses-cylinders with embryos) support the latter
conclusion i.e. that developmental capacities of embryos with that type of
aberration are dependent on genetic background. This is most likely also the
reason for differences in developmental potencies of tetraploid embryos
obtained by Snow (1973, 1976), and Tarkowski et al (1977).
Influence of genotype on development of triploids even before implantation
was suggested by Braden (1957) who observed lower survival rate and high
mortality of extensively homozygotic triploids in silver-strain mice. Also
McGrath & Hillman (1981) found that dispermic triploids + / + /t12 and
+ /t12/t12 developed normally to the blastocyst stage, but triploid t12/t12/t12 are
inviable.
Experimentally induced triploids described in the present study show phenotypic anomalies similar to those found in spontaneously arising embryos. The
method described yields about 40 % of implanted triploid embryos, which is
88
A. NIEMIERKO
10 times the rate of spontaneous appearance of such embryos. It offers possibility
of mass production of triploids for experiments on preimplantation embryos
which may be crucial importance in solving the question of their abnormal
development.
I wish to express my gratitude to Professor A. K. Tarkowski for his advice and guidance
in the course of present work. A special thank you to Dr Jolanta Karasiewicz for comments
on the manuscript.
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