/ . Embryo/, exp. Morp/i. Vol. 52, pp. 1-11, 1979
Printed in Great Britain (g Company of Biologists Limited 1979
Ultrastructural studies of lethal
mouse embryos
lc25H
2SH
c
By MARY D. NADIJCKA, 1 NINA HILLMAN 1 AND
SALOME GLUECKSOHN-WAELSCH 2
From the Department of Biology, Temple University, Philadelphia,
and the Department of Genetics, Albert Einstein College of Medicine,
New York
SUMMARY
Mouse embryos which are homozygous for the c25H deletion at the albino locus are
developmentally arrested at the 2- to 6-cell cleavage stages. This study reveals that the mutant
embryos cease development before they can be distinguished ultrastructurally from their
normal litter-mates. After prolonged developmental delay (24-48 h), the nuclei of the
mutant embryos become extremely aberrant in shape, whereas other subcellular organelles
remain normal and there are no signs of pyknosis. Eventually, the mutant embryos do become
pyknotic and begin to degenerate. The striking effects of this deletion on nuclear ultrastructure
of cleavage-stage embryos are discussed in relation to biochemical and ultrastructural defects
caused by other lethal deletions at the albino locus.
INTRODUCTION
Six radiation-induced mutations at the albino locus (chromosome 7) in the
mouse have been found to be deletions and homozygous lethals (Erickson,
Gluecksohn-Waelsch & Cori, 1968; Erickson, Eicher & Gluecksohn-Waelsch,
1974; Gluecksohn-Waelsch, Schiffman, Thorndike & Cori, 1974; Miller et al.
1974; Jagiello et al. 1976). The longest of the deletions, c25FI, is the earliest acting
lethal, killing during early embryogenesis (Gluecksohn-Waelsch et al. 1974).
Recently, Lewis (1978) found that the homozygous c25H embryos were developmentally arrested at the 3- to 6-cell cleavage stages and that their blastomeres,
at the level of light microscopy, appeared to be bi- or multi-nucleated. She
suggested, therefore, that the lethal phenotype may include aberrant karyokinesis and/or cytokinesis. The results of an ultrastructural study of the
morphological effects of the c2m allele which is reported here show that the
homozygous mutant embryos have abnormally shaped nuclei rather than bior multi-nucleated cells. The ultrastructure of other cellular components is not
affected by the mutant genotype.
1
Authors' address: Department of Biology, Temple University, Philadelphia, Pennsylvania 19122, U.S.A.
2
Author's address: Department of Genetics, Albert Einstein College of Medicine, Bronx
New York, 10461, U.S.A.
2
M. D. N A D I J C K A AND OTHERS
MATERIALS AND METHODS
Animals heterozygous for albino (c) and the c2UL allele (c/c257/) were obtained
from the following mating: c/c (AKR albino) x cc7i/c25// (dilute chinchilla).
Offspring carrying the mutant allele {c/c2bH) are distinguishable as albinos from
their litter-mates {c/cch) which are dilute chinchilla. In order to obtain litters
containing homozygous lethal embryos, c/c25H animals were mated inter se.
Control litters were obtained from the following matings: c/cchxc/cch
or
c/c25H x c/cc7t.
Females were time-ovulated by means of intraperitoneal injections of 2-5 i.u.
pregnant mare serum (Gestyl, Organon) followed 45 h later by 2-5 i.u. human
chorionic gonadotropin (Pregnyl, Organon). After the second injection, females
were mated to the genetically appropriate males and copulation plugs were
checked the next morning (day 0). All embryonic ages were recorded postovulation. Ovulation was presumed to have occurred 12 h after the injection of
human chorionic gonadotropin. The approximate age of the embryos removed
from the female or from culture at specific stages are: late 2-cell, 36 h; late
4-cell, 54 h; 8-cell, 66 h; early morula, 76 h; late morula, 88 h; blastocyst, 98 h.
In the first series of studies, 2-cell embryos were removed from experimental
and control females, placed into standard egg culture medium (Goldstein,
Spindle & Pedersen, 1975) and cultured according to the technique of Brinster
(1963). They were allowed to develop in vitro until the embryos from control
crosses and normal control littermates in experimental crosses had reached the
blastocyst stage. A comparison of the numbers of embryos developmentally
arrested and of the stages of developmental arrest in control and experimental litters was used to verify the lethal nature of the mutation as well as the
phenocritical period for its expression in homozygotes.
In the second series, experimental and control litters were again obtained at
the 2-cell stage. Some litters from both groups were fixed immediately. Others
were placed into standard egg culture medium and allowed to develop in vitro.
At the late 4-cell stage, all of the developmentally retarded embryos (the
putative c2bII/c2511 embryos) from the experimental litters as well as the
developmentally delayed embryos from the control litters were removed from
culture and fixed. The remaining viable embryos of the experimental and control
litters were allowed to develop to the late 8-cell stage. At this point, all embryos
which had failed to advance in development from the 4-cell stage were removed
and examined at the ultrastructural level. The embryos which continued
development were allowed to remain in culture until they reached either the
morula or blastocyst stages when they werefixedand examined for abnormalities.
Using this protocol, all of the developmentally arrested embryos were processed
for study within a short time period after cessation of development.
Two parallel experiments were done in the third series of studies. In the first,
experimental and control litters were removed at the 2-cell stage and separately
Studies o/c 2 5 H /c 2 5 H embryos
3
placed into culture. At each subsequent stage of development, entire experimental and control litters were removed and fixed for microscopic examination.
In the second study, entire experimental and control litters were removed from
females at the 2-cell, 4-cell, 8-cell, early morula and blastocyst stages and fixed
immediately. The ultrastructure of the developmentally arrested mutant embryos
from the younger litters was compared with that of developmentally arrested
embryos obtained from older litters. This comparison enabled us to assess the
ultrastructural effects of the developmental arrest over progressively longer
periods of time (ranging from 12 to 60 h). The two sets of studies in this series
also allowed us to determine if there were differences between the in vivo and
in vitro expression of the mutant genome.
Both experimental and control embryos were fixed in 3 % glutaraldehyde in
0 1 M - P 0 4 buffer (pH 7-4) for 1 h. The embryos were then washed in 0-1 M - P 0 4
buffer overnight, postfixed in 1 % osmium tetroxide (Millonig's, pH 7-3),
dehydrated through a series of alcohol concentrations and embedded in Epon.
Serial sections were stained either with lead citrate for 2 min (Venable &
Coggeshall, 1965) or with saturated uranyl acetate (Watson, 1958) for l h
followed by lead citrate for 2 min. The sections were viewed with a Philips 300
electron microscope. At least one litter in each of the control and experimental
series was stained with 0-5% aqueous uranyl acetate before dehydration in
order to identify putative viral particles CChase & Piko, 1973). These latter
embryos were then dehydrated, embedded and sectioned. The sections were
stained with both uranyl acetate (1 h) and lead citrate (2 min).
Descriptions of the ultrastructure of normal cleavage-staged mouse embryos
are available (Calarco & Brown, 1969; Hillman & Tasca, 1969). Therefore, this
report includes descriptions of only those structures of the mutant embryos
which differ morphologically from those of control embryos or those which
become morphologically altered after the mutant embryos have been in developmental arrest for progressively longer periods of time.
RESULTS
Series 1
The results from the first series of experiments are presented in Table 1.
The data show that 96 % of the control embryos developed from the 2-cell to
the blastocyst stage in vitro. The finding that approximately one fourth of the
experimental embryos die during the early cleavage stages confirms the
observation by Lewis (1978) that the c25J/ deletion acts as an early lethal in
homozygotes. Table 1 also shows that the developmental arrest of the putative
c 25/J /c 25// embryos occurs from the 2- to 6-cell stages, with the greatest number
ceasing development at the 4- to 6-cell stages.
The same frequency of arrested and probably homozygous mutant embryos
has been observed in experimental litters developing both in vivo and in vitro. In
M. D. NADIJCKA AND OTHERS
Table 1. Stage of developmental arrest in vitro'
Experimental
Control
Total embryos
464
316
Arrested embryos
18(4)
91 (29)t
Stage of arrest
2-cell
4 (0-9)
13 (4)t
3-cell
4 (0-9)
12(4)t
3 (0-6)
4-cell
29 (9)t
5-cell \
2 (0-4)
37 (12)t
6-cell /
1 (0-2)
8-cell
0 —
Early morula
0 —
4 (0-9)
Late morula
0 —
0 —
Blastocyst
0 —
0 —
* Embryos kept in culture medium from the 2-cell to blastocyst stage.
t A significantly higher number of embryos were developmentally arrested in the
experimental litters (P < 001) than in the control litters. Significance was determined by the
contingency chi square.
addition, the phenocritical period for the mutant embryos developing in vivo
corresponds to the in vitro range of developmental arrest. No ultrastructural
differences have been found between the embryos of the in vitro and in vivo
experimental series. For this reason, the two groups of mutant embryos are not
distinguished in this report.
Series 2
Comparative ultrastructural observations at the 2-cell cleavage stage of 25
embryos from the control matings and 20 embryos from experimental matings
show that the mutant embryos cannot be distinguished from either their correspondingly staged litter-mates or control embryos at this stage of development.
All of the embryos contain cellular organelles which are structurally normal for
this early cleavage stage.
Additional experimental and control litters were allowed to continue development beyond the 2-cell cleavage stage. Of the 196 experimental embryos which
were examined at the late 4-cell cleavage stage, 14 embryos had not advanced
beyond the 2-cell stage and 11 remained as 3-cell embryos. Among the corresponding 216 control embryos, only three embryos did not advance to the
4-cell stage. Of the 171, 4-cell experimental embryos, reincubated for a time
sufficient for them to attain the 8-cell stage, 18 remained as 4-cell embryos and
17 were developmentally delayed as 5- and 6-cell embryos. Only three of the
control embryos failed to advance from the 4-cell stage to the 8-cell stage. All
60 developmentally retarded embryos from experimental litters were examined
ultrastructurally. With a few exceptions (two 2-cell, two 4-cell and three 6-cell)
Studies o/c 2 5 H /c 2 5 H embryos
5
the developmentally delayed experimental embryos were structurally normal.
The exceptional embryos contained aberrantly shaped nuclei. However, since
so few of the control embryos in these early cleavage stage litters show an
aberrant structure, the only criterion for a tentative classification of homozygous
mutant embryos is retardation of development. An examination of the six
developmentally retarded embryos from control crosses revealed that none
contained aberrantly shaped nuclei.
All of the remaining experimental and control embryos continued to develop
to morula and blastocyst stages and all were structurally normal. As in the
studies of Lewis (1978) there are no indications that the deletion affects either
the morphology or viability of c/cZ5H preimplantation embryos.
Series 3
Consistent ultrastructural abnormalities are found only after the abnormal
embryos from experimental litters have been in developmental arrest for an
extended period of time. For example, an 8-cell experimental litter contains
developmentally arrested embryos ranging from the 2- to 6-cell stages. While
most of the 4-, 5- and 6-cell embryos appear structurally normal, the 2- and
3-cell embryos which have been in developmental arrest for a longer period of
time contain irregularly shaped nuclei. Three of the four 2-cell embryos and all
four of the 3-cell embryos were examined ultrastructurally by serial sections
and found to contain nuclei with aberrant shapes. The degree of the aberrancy
varies from cell to cell both within and between embryos. While some nuclei
are only moderately misshapen, e.g. crescent-shaped or elongated, others are
more abnormal in configuration (Figs. 1, 2). Although the examination of
randomly selected sections suggests that cells may be bi- or multi-nucleated,
a study of serial sections reveals that the apparently separate nuclear lobes are
either attached to each other or to larger nuclear areas. None of the developmentally arrested embryos contain binucleated or multinucleated cells. All
13 of the developmentally arrested embryos from control animals have been
studied. None of them contain aberrantly shaped nuclei.
Twenty-six of the arrested 2- to 6-cell embryos obtained from experimental
litters at the early morula stage have the same abnormally shaped nuclei as those
noted in the youngest, developmentally arrested mutant embryos from litters
in the 8-cell stage. The morphology of other organelles is not adversely affected
and none of the putative mutant embryos exhibit degenerative effects from the
extended time in arrest. Conversely, developmentally arrested embryos from
control litters are in varying stages of degeneration.
Developmentally retarded embryos in experimental litters examined at the
late blastocyst stage present a range of degenerative defects. Some show no signs
of degeneration while others are totally pyknotic. Those with the lowest grade
of degeneration (a total of 28 embryos) have nuclei which are multilobed and
have narrow fingerlike projections (Fig. 3). When these cells, which appear to
M. D. N A D I J C K A AND OTHERS
Fig. 1. Section of a developmentally arrested 2-cell embryo from an experimental
litter at the 8-cell stage. Note the horseshoe-shaped nucleus. Primary nucleoli (P)
and cytoplasmic organelles appear normal, x 6800.
Fig. 2. Section of part of an arrested embryo in the 3-cell stage from an experimental litter at the 8-cell stage. Note the aberrantly shaped nucleus, x 6500.
Studies of c 25H /c 25H embryos
Fig. 3. Section of a developmentally arrested embryo in the 2-cell stage from an
experimental litter in the blastocyst stage. Note the nuclear projections and
locations, x 6600.
be multinucleated in single sections, are traced serially, small bridges of nuclear
material are found connecting the almost severed nuclear lobes to the main
body of the nucleus. No binucleated or multinucleated cells have been observed.
Other cellular organelles are not affected by the prolonged period of
developmental arrest.
The remainder of the arrested embryos contain both pyknotic nuclei and
pyknotic cells in varying numbers. In the most degenerated state, the plasma
and nuclear membranes are discontinuous and organelles are indistinct. The
small number of control embryos which are developmentally arrested at the
2- to 6-cell stages and which have been observed when their litter-mates are in
the early morula or blastocyst stages, are either completely pyknotic or totally
decomposed.
Virus observations
Viral aggregates, judged to be A particles by both their size and perinuclear
location (Biczysko, Solter, Graham & Koprowski, 1974; Solter, personal
communication) have been found in both experimental and control litters at
the 2-cell stage (Fig. 4A, B). In control embryos and in the normal litter-mates
of the mutant embryos, these viral aggregates disperse after the 2-cell stage. In
later stages, single viruses are randomly distributed in the cytoplasm. Among the
M. D. NADIJCKA AND OTHERS
_
. _. . . _
.
: *
Fig. 4. (A) Portion of developmentally arrested 2-cell embryo from an experimental,
litter in morula stage. Note perinuclear clusters of viral particles, x 8400. (B) A
higher magnification of a type A virus particle found in both control and experimental embryos, x 130000.
arrested embryos, removed from litters in 4-cell, 8-cell, early morula and blastocyst stages, all of the 2-cell and a few of the 3- and 4-cell embryos retain their
perinuclear viral clusters. In the remainder of the 3- and 4-cell embryos and in
all mutant embryos in later stages the viruses are dispersed in the cytoplasm.
DISCUSSION
Of the six radiation-induced deletions at the albino locus in the mouse, two
are lethal prenatally (cm, c25H) and four perinatally (c14Cos, cm, c65K, c112K).
The perinatal syndrome covers a multiplicity of biochemical and morphological
effects. The biochemical abnormalities include deficiencies in the activities of
glucose-6-phosphatase, tyrosine aminotransferase, serine dehydratase (Gluecksohn-Waelsch et ah 1974), glutamine synthetase (Gluecksohn-Waelsch,
Schiffman & Moscona, 1975) and UDP-glucuronyltransferase (Thaler, Erickson
Studies o/c 2 5 H /c 2 5 H embryos
9
& Pelger, 1976). The mutant neonates also have decreased concentrations of
albumin, a fetoprotein, and transferrin, the principal plasma proteins (Garland,
Satrustegui, Gluecksohn-Waelsch & Cori, 1976).
Morphologically, the perinatal mutants can be distinguished from their
litter-mates by the abnormal ultrastructure of specific subcellular membrane
systems in kidney and liver parenchymal cells. The affected cells have dilated
and vesiculated rough endoplasmic reticulum, dilated Golgi apparatus and
abnormal nuclear membranes (Trigg & Gluecksohn-Waelsch, 1973). Neonatal
litter-mates heterozygous for the mutant lethal alleles are both biochemically
and morphologically normal (Gluecksohn-Waelsch & Cori, 1970; Trigg &
Gluecksohn-Waelsch, 1973).
There have been no previous comparative biochemical or ultrastructural
studies of embryos homozygous for either of the prenatal mutations, cGI[ and
c25H. However, enzyme and serum protein studies have been reported of
complementing genotypes heterozygous for ceH or c 25// and one of the perinatal
lethal mutations (Gluecksohn-Waelsch et al. 1974). Morphological studies of
CM/C<ur embryos have been limited to a light microscopic examination of the
embryos during the phenocritical and lethal periods. They can be distinguished
from their phenotypically normal litter-mates at 6-5-7 days of gestation by
their severely reduced size and by abnormalities of the ectoplacental cone and
parietal endoderm (Lewis, Turchin & Gluecksohn-Waelsch, 1976).
The albino deletion described in this report (c25//) causes developmental
arrest of homozygotes prior to the second, or during the third, cleavage division
of the fertilized ovum. It is, therefore, the earliest acting lethal mutation
reported in mammalian embryos. c 25// is the largest of the albino deletions and in
contrast to cm fails to complement fully with any of the other deletions at the
albino locus (Gluecksohn-Waelsch et al. 1974). However, c25H, like the other
five lethal deletions, acts as a true recessive.
The ultrastructure of the homozygous c2bil embryos resembles that of their
litter-mates and of other control embryos when these mutant embryos are
examined soon after developmental arrest. Such embryos appear to be viable
and non-necrotic both in vitro and in vivo until their litter-mates reach the
blastocyst stage. Bizarrely shaped nuclei are not found until the embryos have
been in developmental arrest for some time. It is of interest to note that the
C257/ deletion, unlike the perinatal lethal deletions, does not appear to affect the
subcellular membrane system of cleavage stage embryos. Membranes retain
their ultrastructural integrity until the embryos are in advanced stages of
degeneration. Nevertheless, the effect of this deletion on membranes cannot be
entirely ruled out since complementing genotypes of c25H and one of the perinatal lethals show ultrastructural defects of rough endoplasmic reticulum, Golgi
apparatus and nuclear membrane (Gluecksohn-Waelsch et al. 1974), and the
aberrant nuclear configuration of c25H homozygotes could result from a defect
of the nuclear envelope. Also, because of the paucity of both rough endoplasmic
10
M. D. NADIJCKA AND OTHERS
reticulum and Golgi apparatus in the early embryonic stages, subtle effects
would not be necessarily observable. Our studies, however, have failed to show
bi- or multi-nucleated cells in the arrested, and presumably homozygous mutant
embryos.
In our studies the phenocritical period of c25H homozygotes occurs at the
2- to 6-cell stage. Lewis (1978) reported it to be between the 3- and 6-cell stages.
This minor discrepancy may be due to differences of genetic backgrounds used
in the two studies. Whereas Lewis used heterozygous parents from a brothersister strain inbred for eight to nine generations, we obtained heterozygous
c/c25//animals from outcrosses of cc"/c25H to albino AKR.
The AKR genetic background may also account for the viral aggregates
which we observed in both control and experimental litters and which bear no
relation to the presence or absence of c25H. Biczysko, Pienkowski, Solter &
Koprowski (1973) compared the concentration of A particles in blastocysts
obtained from eight different strains of mice. They found AKR embryos to
harbour the highest concentration of A-type particles, and suggested that
A particle replication and maturation may be expressed in only specific strains
of mice, the AKR background genotype being one which supports this
expression.
This research was supported by United States Public Health Service Grants HD 00827,
GM 19100 and HD 00193, and by a grant from the American Cancer Society VC-64. The
authors would like to thank Marie Morris and Geraldine Wileman for their technical
assistance.
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25//
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{Received 1 November 1978, revised 3 January 1979)