/. Embryol. exp. Morph. Vol. 69, pp. 223-236,1982
223
Printed in Great Britain © Company of Biologists Limited 1982
The development of monosomy 19 mouse embryos
By TERRY MAGNUSON,1 SANDRA SMITH AND
CHARLES J. EPSTEIN
From the Departments of Pediatrics and of
Biochemistry and Biophysics, University of California, San Francisco
SUMMARY
In general, autosomal monosomy is lethal much earlier in mammalian development than
autosomal trisomy. In an attempt to understand why monosomy is so deleterious, we have
begun to characterize the development of mouse embryos monosomic for chromosome 19.
A dramatic loss of monosomy 19 embryos was found to occur between days 3 and 4 of
development. This loss occurred both in vivo and in vitro and with intact blastocysts or
isolated inner cell masses. Experiments with inbred strains showed that this loss was not;
due to the expression of recessive lethal genes. While monosomic embryos were found to
have fewer cells than normal and trisomic litter-mates beginning at the early morula stage,
the ability to form blastocysts is not interfered with. Electron microscopy revealed no differ*
ence in the cellular ultrastructure of monosomic when compared with diploid embryos.
Furthermore, two-dimensional gel electrophoresis did not reveal any differences in the
proteins synthesized by monosomic, trisomic or diploid litter-mates when examined at day 3
of development. These results indicate a lack of gross genomic disturbances in monosomic
embryos. When monosomy<-> diploid chimaeras were made, viable monosomic cells were
found in day-9 post-implantation embryos, well past the lethal period. Thus, in chimaeric
embryos, the normal cells appear to be able to provide whatever is lacking, suggesting that
monosomy 19 is not a cell lethal. Instead, death may be due to a dosage alteration in specific
gene products needed during early development.
INTRODUCTION
When aneuploid progeny are produced in mice by breeding techniques
that result in a high rate of non-disjunction, trisomic embryos are found
to survive well past implantation (White, Tjio, Vandewater & Crandall, 1974;
Gropp, Kolbus & Giers, 1975). In contrast, monosomic embryos generally dfe
before or during implantation (Dyban & Baranov, 1978; Epstein & Travis,
1979; reviewed by Magnuson and Epstein, 1981). Studies in man on spontaneous
abortions also reveal that while a large number of trisomic foetuses are identified,
monosomic foetuses are rarely found (Boue, Boue & Lazar, 1975; Hassold et 0,1.
1978). Therefore, in attempting to further our understanding of the pathogeneSis
of conditions resulting from aneuploidy, it is of interest to determine why
autosomal monosomy is lethal so much earlier in development than is trisomy.
1
Author's address: Room 1421 HSW, Department of Pediatrics, University of California,
San Francisco, California 94143, U.S.A.
8-2
224
T. MAGNUSON, S. SMITH AND C. J. EPSTEIN
/ \
/ \ -f
/ \
\ J 19
X
/ \ 5
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f\
f*\
*• Monosomy 19
v /9
^
i
1 9
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/ 1199
A 19 V
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i y
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Rb(919)163H/Rb(519)lWh
doubly heterozygous male
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Trisomy 19
Fig. 1. Production of mouse embryos aneuploid from chromosome 19. When a
mouse is doubly heterozygous for two different Robertsonian metacentric chromosomes, each of which has a chromosome in common, non-disjunction occurs during
meiosis about 20 % of the time. Thus, when a male carrying two of these metacentric
chromosomes is mated with a normal female, the resulting litter will consist of
monosomic, trisomic and diploid embryos.
In an earlier study (Epstein & Travis, 1979) using males carrying both the
Rb(8.19)lCt and Rb(9.19)163H translocations as fathers, we observed that
mouse embryos monosomic for chromosome 19 have a very well-defined time
of death. The frequencies of trisomy and monosomy are equal (approximately
15-20 % each) on day 3 (early blastocyst) of preimplantation development, but
on day 4 (late blastocyst), the frequency of monosomic embryos decreases
greatly while that of the trisomic embryos remains the same. We have now
repeated these studies using males carrying a different combination of translocation chromosomes and have obtained similar results. We have also extended
these studies in an attempt to begin to understand why monosomy embryos die
so early in development. Our results suggest that monosomy 19 is not a cell
lethal, and that death may be due directly to the reduction by half of the synthesis of specific gene product(s) needed at that time in development.
MATERIALS AND METHODS
A. Breeding scheme for the production of aneuploid embryos. Male mice
homozygous for the Rb(5.19)lWh Robertsonian translocation chromosome
(obtained from Dr D. Trasler, McGill University, Montreal, Canada) were
mated with females homozygous for the Rb(9.19)163H Robertsonian translocation (obtained from Dr A. Gropp, Abteilung fur Pathologie der Medizini-
Aneuploid mouse embryos
22$
Fig. 2. Identical twin half embryos obtained by the twin embryo technique. The
two blastomeres of a 2-cell embryo were separated from one another as described
in the text. Each half embryo was individually cultured for 4 days. One twin embryo
was then spread (A) allowing for karyotypic identification. Once identified, the
other twin (B) was used for experimental purposes. Magnification: B = 400 x .
schen Hochschule Liibeck, Germany). To obtain monosomy 19, trisomy 19 and
diploid embryo litter-mates, the doubly heterozygous Rb(5.19)lWh/Rb(9.19)
163H males were mated with superovulated ICR Swiss Albino or C57BL/6
females (obtained from Simonsen Laboratories, Gilroy, California) (see Fig. 1
for diagram of breeding scheme). Standard procedures were then used to flush
embryos from oviducts or uteri with modified Whitten's medium (Epstein,
Wegienka & Smith, 1969; Golbus & Epstein, 1974). The day the fertilization
plug is observed is designated as day 0.
B. Karyotyping of preimplantation embryos. Embryos were first incubated
for 3-6 h in medium containing 0-07 /tg/ml vinblastine (Velban, Gibco). After
incubation, the embryos were placed in 0-75 % sodium citrate for 2 min &nd
then transferred to a 3:1 methanol:acetic acid fixative for an additional 2 min.
Each fixed embryo was then transferred to a pre-cleaned microscope slide, and
226
T. MAGNUSON, S. SMITH AND C. J. EPSTEIN
Day 1
Day 2
C57 BL/6 X Rb(9-19) 163H/Rb(5-19) 1 Wh(GPIb)
Culture
Day 9
Karyotype
Fig. 3. Preparation and characterization of aneuploidy 19*-> normal chimaera.
a microdrop of a 1:1 acetic acid: 50 % lactic acid solution was placed on the
embryo. When the embryo was observed to break apart, it was spread on the
slide with additional methanol: acetic acid fixative. The slide was dried for a
minimum of 15 min on a 37 °C warming plate and then stained with 3 % Giesma
(Gurr) for 8-5 min. Monosomic embryos have 39 acrocentric chromosomes,
while trisomic embryos have 37 acrocentric and 2 metacentric chromosomes and
diploid embryos have 38 acrocentric and 1 metacentric chromosome.
C. Twin embryo technique. The zona pellucida was removed from 2-cell
embryos with 0-5 % pronase (Calbiochem) in phosphate-buffered saline (Mintz,
1962). The embryos were then washed five times in medium. The two blastomeres of an individual embryo were separated from one another by gentle
aspiration in a micropipette (Epstein, Smith, Travis & Tucker, 1978). The pairs
of blastomeres were then transferred to wells of a Terasaki microtest tissue
culture plate (Falcon 3034) and cultured at 37 °C in 5 % CO2 in air under oil
(10 ml Fisher light paraffin oil per plate). On the morning of the fourth day of
culture, one twin was karyotyped (Fig. 2 a) while the other twin (Fig. 2 b) was
used for experimental purposes.
D. Two-dimensional (2D) electrophoresis. Monosomic, trisomic or diploid
twin embryos at the early to mid blastocyst stage were grouped into pools of
6 and were then incubated for 4 h in medium containing approximately 2
mCi/ml [35S]methionine (specific activity of 900-1100 Ci/mmole, Amersham)
(Magnuson & Epstein, 1981 b). After incubation the embryos were washed three
times with phosphate-buffered saline and transferred to a glass test tube. The
excess phosphate-bufTered saline was removed with a micropipette and the
embryos were solubilized in 20/d of O'Farrell's (1975) isoelectric focusing
227
Aneuploid mouse embryos
Table 1. Survival of early mouse embryos aneuploid for chromosome 19*
Cross: ICRx Rb(9 J9)163H/Rb{5.19)1 Wh
%j of total> embryos
Stage
Sample size
Monosomic
Trisomic
Diploid
8- to 16-cell
Morula
Early blastocyst
Late blastocyst
Day 3 to day 4
(in vitro)
Day 3 ICMf to
day 5 ICM
(in vitro)
Day 9%
132
102
122
77
16
11
17
3
21
13
17
17
63
76
56
80
161
7
22
71
58
61
3
0
17
21
80
79
* Embryos were flushed at appropriate stage and then karyotyped.
t ICM = inner cell mass, isolated by immunosurgery (Solter & Knowles, 1975).
$ Cross: C57BL/6>c Rb(9.19)163H/Rb(5.19)lWH.
sample buffer. Two-dimensional gel electrophoresis was performed as outlined
by O'Farrell (1975) with a 4-5% stacking gel and a 10% separating gel being
used in the second dimension. Kodak NS-2T film was used for autoradiography of the dried gels.
E. Electron microscopy. Twin embryos at the early to mid-blastocyst stage
were prepared for thin-section electron microscopy as previously described
(Magnuson, Demsey & Stackpole, 1977). The stained sections were examined
with a Hitachi HS-8 electron microscope.
F. Preparation of aneuploidy 19 chimaeras. Aggregation chimaeras were made
by the general method described by Mintz (1971) (see Fig. 3 for diagram).
Briefly, superovulated C57BL/6 females were mated with Rb(9.19)163H/
Rb(5.19)lWh males to produce monosomic, trisomic or diploid embryo
litter-mates that were glucose phosphate isomerase-1 type b (GPP). The embryos
were removed from the mother at the 2-cell stage and cultured overnight Until
the 4-cell stage, at which point the zona pellucida was removed. These embryos
were then aggregated with 8- to 16-cell BALB/c embryos (GPIa) in a 1:100
dilution of phytohaemagglutinin (PHA-P, Difco) in phosphate-buffered saline.
The aggregates were washed and cultured overnight until the late morula to
early blastocyst stage. Then, approximately 10-12 embryos were implanted With
a micropipette into each uterine horn of pseudopregnant ICR females. The
pregnant females were killed 7 days later and the embryos dissected out.
G. Determination of chimaerism. The existence of chimaerism was assessed
by analysis of the GPI pattern. Portions of the head and tail and a portion of
228
T. MAGNUSON, S. SMITH AND C. J. EPSTEIN
Table 2. Mean cell number per embryo*
Stage
8- to 16-cell
Morula
Early blastocyst
Monosomic
Trisomic
Diploid
13±2t
15±2J
46±3J
13±2
12±2
60±3
12±2
22±2
56±2
• Cell counts were done on the same population of embryos used in Table 1.
t ±SEM.
X Significantly different from normal and trisomic littermates, P < 0005.
the extraembryonic membranes of each embryo were placed in separate aliquots
of 0-04 M-Tris-glycine, pH 8-6, and vigorously aspirated. After freeze-thawing
three times, 3-5 fi\ of the extract was applied to a cellulose acetate (Cellogel,
Chemetron) sheet and electrophoresed in the Tris-glycine buffer at 250 V
(constant current) for 3-5 h. The GPI bands were stained as described by Van
Someren et al. (1974).
For karyotyping, the remaining portions of each embryo and membranes
were incubated for 6 h in Dulbecco's modified Eagle's medium with 10%
foetal calf serum, penicillin and streptomyocin, and vinblastine (007/ig/ml).
After the incubation, each sample was treated with 0-56% KC1 for 15 min at
37 °C,fixedin 1 ml of 3:1 methanol:acetic acid for 15 min and then transferred
to 0*1 ml 60% acetic acid. After 5 min, the acetic acid solution was transferred
to a slide pre-warmed to 50 °C. After drying, the slides were stained with
Giemsa.
RESULTS
A. Survival of embryos aneuploid for chromosome 19
Approximately equal frequencies of monosomic and trisomic embryos were
found at the 8- to 16-cell, morula and early blastocyst (day 3) stages (Table 1,
lines 1-3). However, at the late blastocyst stage (day 4), a considerable drop in
the percentage of embryos that were monosomic was found (Table 1, line 4).
In contrast, the percentage of trisomic embryos remained the same. At day 9,
while the expected frequency of trisomic embryos was present, no monosomic
Fig. 4. Two-dimensional gel autoradiograms of proteins synthesized by monosomic
(A), trisomic (B) and diploid (C) litter-mates. No consistent difference was detected
in the proteins synthesized by the three types of embryos. Separation from left to
right is by charge with a pH gradient ranging from 6-8 (on the left) to 4-6 (on the
right). Separation from top to bottom is by size. For A, 65000 cpm were loaded
on to the gel and exposure time was 83 days. For B, 67000 cpm were loaded and
exposure time was 83 days. For C, 95000 cpm were loaded and gel was exposed for
90 days. A, Actin proteins, T, tubulin proteins.
Aneuploid mouse embryos
4C
229
230
T. MAGNUSON, S. SMITH AND C. J. EPSTEIN
Aneuploid mouse embryos
231
GPI
Fig. 6. Glucose phosphate isomerase (GPI) patterns of monosomy <-> diploid (M-D)
and diploid •-» diploid (D-D) litter-mate chimaeras. The monosomic or diploid
metacentric component of the chimera is marked by GPIb and the normal diploid
by GPP. C, control mixture of GPP and GPP.
embryos were found (Table 1, line 7). This loss of monosomic embryos occurred
both in vivo (Table 1, line 4) and in vitro (Table 1, line 5) and with both intact
blastocysts and isolated inner cell masses (Table 1, line 6). Since the chromosome 19 which is present in monosomic embryos comes from the mother (see
Fig. 1), experiments with C57BL/6 mothers show that this loss of monosomy 19
embryos is not due to the expression of recessive lethal genes (Table 1, line 7;
see also data from Epstein & Travis, 1979).
While the ability to form blastocysts is not interfered with and, on a gross
morphological level, monosomy 19 embryos appear normal until the time of
death, we have found that, beginning at the morula stage, monosomic embryos
have fewer cells than normal and trisomic litter-mates (Table 2). This decrease
in cell number was also found in early blastocysts.
B. 2D gel electrophoresis
A comparison of about 500 proteins synthesized by monosomic, trisomic or
diploid twin embryos was made using 2D gel electrophoresis and autoradiography. Approximately equal numbers of radioactive counts were loaded on to
each gel which was then exposed to X-ray film for about the same amount of
time. When the autoradiograms were examined, no consistent difference was
found in the proteins synthesized by the three types of embryos (Fig. 4).
Fig. 5. Thin-section electron micrographs of inner-cell-mass cells from a monosomic
(A) and diploid (B) embryo. Arrows, degradative bodies; arrowheads, mitochondria; L, lipid droplets; G, Golgi apparatus; E, endoplasmic reticulum. Magnification: A, 44 370 x, B, 47430 x .
232
T. MAGNUSON, S. SMITH AND C. J. EPSTEIN
7A
70
7B
7E
7F
7C
Fig. 7. Light micrographs of a monosomy<-» diploid (A) and a diploid<-> diploid (D)
chimaera. For the monosomy*-* diploid chimaera, the monosomic karyotype (39
chromosomes) is shown in B and the diploid karyotype (40 chromosomes) is shown
in C. For the diploid <-> diploid chimaera, the diploid metacentric karoytype is shown
in E (38 acrocentric and 1 metacentric chromosome, the metacentric being indicated
by the arrow) and the diploid normal karyotype (40 chromosomes) is shown in F.
Magnification, A and B: 60 x.
C. Cellular ultrastructure
When examined by thin-section electron microscopy, monosomic twin
embryos appeared to be at the same developmental stage as their diploid
litter-mates (Fig. 5). No difference in the ultrastructure of the nucleus, nucleoli,
mitochondria, endoplasmic reticulum, Golgi apparatus, polysomes or inter-
Anneploid mouse embryos
233
Table 3. Composition ofchimaeric embryos*
Number ofchimaeric embryos
RblWh/Rbl63H r
input
M19*-»2N
T19<->2N
2N<->2N
<50%
>50%
5
0
2
5
2
28
* Determined either by counting chromosome spreads or by estimating relative intensities
of GPI bands. These determinations were not made on all of the chimaeras.
cellular junctions was found when three monosomic and three diploid embryos
were analysed. The ultrastructure of these organelles appeared similar to that
described by Calarco & Brown (1969) as being normal for blastocysts. The
only monosomy 19-associated defect observed was an increase in the number of
degradative bodies in the cytoplasm of monosomic embryos when compared to
diploid embryos. When 21 random sections from three monosomic embryos
were examined and compared to an equal number of sections from three normal
embryos, 3-18 ±0-69 (SEM) degradative bodies per section were found in the
monosomic embryos and 1-85 ±0-4 per section were found in normal embryos
(P < 005).
D. Analysis of aneuploidy 19 chimaeras
A total of 744 aggregates were made and transferred into foster mother$. Of
these, 90 (12%) implanted and grew. When dissected at day 9, 74 (82%) of the
implants were chimaeric as determined by GPI analysis (Fig. 6). Ten were
chimaeric in the extraembryonic membranes only while 34 were chimaefic in
both membranes and embryos. The remaining 30 were chimaeric but embryos
and membranes were not separated. Of the 74 implants, 69 were succes$fully
karyotyped. Five (7 %) of the chimaeras were monosomy 19 <-> diploid. Of these,
three were chimaeric in both embryo and extraembryonic membranes and two
were chimaeric but embryo and membranes were not separated. Nine (13%)
of the chimaeras were trisomy 19 <-> diploid. Of these, four were chimaeric in
both embryo and membranes, two were chimaeric in membranes only and three
were chimaeric but embryo and membranes were not separated. All of the
monosomy 19 <-> diploid chimaeras appeared as healthy day-9 embryos (Fig. 7).
The aneuploid contribution in the chimaeric embryo was always less than 50 %
for the monosomy 19 <-> diploid chimaerias (Table 3). In contrast, the aneuploid
or metacentric contribution in the trisomy <-> diploid and diploid«-»diploid
chimaeras was usually greater than 50 %.
234
T. MAGNUSON, S. SMITH AND C. J. EPSTEIN
DISCUSSION
In an earlier study (Epstein & Travis, 1979), we found that embryos monosomic for chromosome 19 appear to die between days 3 and 4 of in vivo or in
vitro development. We have repeated these studies using a metacentric combination which is different from the combination used in our previous study and
have obtained similar results. In addition to the intact embryo dying, we have
now observed that isolated inner cell masses also die at this time, indicating that
the defect cannot be attributed to the trophectoderm alone. Of the other
aneuploids so far examined, only monosomy 17 embryos die earlier than the
monosomy 19 embryos (Baranov, Dyban & Chebotar, 1980; Magnuson &
Epstein, unpublished results). The others (monosomy 1, 12 or 15, and possibly
5, 9 or 14) die later than day 4 but the exact time of death is not yet known
(Dyban & Baranov, 1978; Epstein & Travis, 1979).
The earliest manifestation of monosomy 19 is retarded cell growth. Beginning
at the early morula stage, the monosomy 19 embryos lag behind in the number
of cells per embryo. No other specific gross or ultrastructural abnormality was
observed except for an increase in the number of degradative bodies found in the
cytoplasm of monosomic embryos. This increase is more likely a manifestation
of a dying embryo rather than a specific defect associated with monosomy 19.
When two-dimensional gel protein patterns of monosomy 19, trisomy 19 and
diploid embryos were examined, no consistent difference was found. Thus, the
cause of death of monosomy 19 embryos remains unknown.
The ability to rescue monosomy 19 embryos in aggregation chimaeras suggests
that the diploid cells are able, for at least some period of time, to provide
whatever is lacking so that aneuploid cells can participate in normal development. This implies that monosomy 19 is not a cell lethal but rather that specific
correctable defect(s) may be associated with this aneuploid condition. These
results, together with our two-dimensional gel data, are compatible with the
idea that monosomy 19 does not result in general genomic disturbances. Instead,
the defect is likely to be due to a 50 % dosage deficiency in chromosome 19 gene
products needed at that time in development.
Previous work indicates that autosomal trisomy also does not result in
widespread alterations in the synthesis, degradation or modification of gene
products coded for by genes not located on the aneuploid chromosome (Weil &
Epstein, 1979; J. Sawicki & C. J. Epstein, unpublished results). A number of
studies do, however, indicate that aneuploidy does result in primary dosage
effects for genes located on the chromosome in question (Krone & Wolf, 1977;
Epstein, Tucker, Travis & Gropp, 1977; Feaster, Kwok & Epstein, 1977;
Kurnit, 1979). Thus, the exact mechanisms by which aneuploidy results in
death or abnormal development are probably due to alterations in specific
cellular processes or structures directly affected by the aneuploid genes. The
important difference between monosomy and trisomy is that monosomy results
Aneuploid mouse embryos
235
in an obligatory state of dosage deficiencies which might then have more
serious consequences on concentration-dependent cellular processes than
trisomy (Epstein, Epstein, Cox & Weil, 1981).
In all cases, the monosomy 19 contribution to the aggregation Chimaeras was
less than 50 %, while the trisomic or diploid metacentric component was usually
much greater than 50%. The decreased monosomic component could be due
to the inability of a chimaera to survive if a majority of the cells are monosomic.
If this were the case, one would expect to find a significant decrease in the
proportion of monosomy 19 «-> diploid chimaeras when compared to the proportion of trisomy 19 <-> diploid chimaeras recovered. While a difference Was
observed, the numbers are too small to determine if it is significant. An alternative explanation for the decreased monosomic component may be that while
monosomy 19 cells are able to survive in a chimaera, they continue to divide at
a slower rate than the diplcid cells and, consequently, are eventually diluted out.
If this were true, the monosomy 19 contribution should be inversely proportional
to the age of the embryo. We are presently testing to see if this is in fact the case.
We would like to thank Ms Teodosia Zamora, Ms Estrella Lamella and Mr Bruce Travis
for excellent technical assistance. T. M. was a recipient of a National Research Service
Award from the National Institutes of Health. This work was supported by NIH grant
GM-24309.
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