/ . Embryol. exp. Morph. Vol. 34, 3, pp. 645-655, 1975
Printed in Great Britain
645
The early development of haploid and aneuploid
parthenogenetic embryos
By MATTHEW H. KAUFMAN 1 AND LEO SACHS2
From the Department of Genetics, Weizmann Institute of Science,
Rehovot, Israel
SUMMARY
The early development of parthenogenetically activated oocytes has been studied in
C57BL x CBA-T6T6 (F1T6) translocation heterozygote mice and C57BL x CBA-LAC
(FXLAC) mice. All FXT6 oocytes had either a quadrivalent or a univalent-trivalent configuration at meiosis I; no such chromosome configurations were observed in the FxLAC
oocytes. At ovulation 36-5 % of the FXT6 oocytes had 19 or 21 chromosomes, whereas 97 %
of the FxLAC had the normal haploid chromosome number of 20. After parthenogenetic
activation, chromosome counts at metaphase of the first cleavage mitosis were made of the
eggs with a single pronucleus following extrusion of the second polar body. These activated
eggs had similar frequencies of 19, 20 and 21 chromosomes as had the oocytes at ovulation.
The activated 1-cell eggs were transferred to the oviducts of pseudopregnant recipients and
the embryos recovered 3 days later. At this stage of development, most of the FiT6 embryos
with 19 chromosomes were no longer found, but the frequency of 21-chromosome embryos
was similar to the frequency of 21-chromosome oocytes and activated eggs. There was a
similar mean number of cells in the embryos with 20 and 21 chromosomes.
The results indicate that nearly all the embryos with 19 chromosomes failed to develop,
probably beyond the 2-cell stage, whereas oocytes with 21 chromosomes had a similar
development to oocytes with 20 chromosomes up to the morula stage.
INTRODUCTION
Spontaneous abortions and cases of congenital malformations in man can
be associated with chromosome abnormalities (Carr, 1971; Fowler & Edwards,
1973), so that it is important to determine the developmental potential of
aneuploid embryos. An abnormal chromosome constitution in the embryo can
be produced by meiotic non-disjunction during gametogenesis, and in normal
diploid animals there is generally an extremely low incidence of this nondisjunction (Beatty, 1970; Rohrborn, 1972; Hulten & Lindsten, 1973; Fowler &
Edwards, 1973). For experiments on aneuploid embryos, it is therefore necessary
to use a system which produces a higher level of chromosome abnormality.
This situation occurs in mice which are translocation heterozygotes (Lyon &
Meredith, 1966; Searle, Ford & Beechey, 1971; Eicher & Green, 1972).
1
Author's address: Anatomy Department, Downing Street, Cambridge CB2 3DY U.K.
Author's address: Department of Genetics, Weizmann Institute of Science, Rehovot,
Israel.
2
41-2
646
M. H. KAUFMAN AND L. SACHS
The T6 translocation involves a reciprocal exchange between segments of
chromosomes 14 and 15 (Miller et al. 1971; Nesbitt & Francke, 1971; Eicher &
Green, 1972). During meiosis in T6/+ mice, the small T6 marker chromosome
(T6M) and large translocation products associate with their two normal
partners either in a quadrivalent or univalent-trivalent configuration, the
univalent being the T6M chromosome (Eicher & Green, 1972; Forejt, 1974).
T6/ + heterozygote mice therefore ovulate both haploid and aneuploid oocytes,
as a result of non-disjunction at meiosis I (Eicher & Green, 1972).
The present experiments were undertaken to determine the effect of aneuploidy on the development of parthenogenetic mouse embryos. Oocytes
ovulated by T6/+ Fx mice, which were expected to have a high frequency of
aneuploidy, were compared with oocytes from non-translocation-bearing F x
mice, which were expected to have a low incidence of aneuploidy. Only the
parthenogenetically activated eggs which developed a single pronucleus following extrusion of the second polar body, and were potentially haploid (Kaufman,
1913a; Graham & Deussen, 1974), were examined. The development of these
embryos was followed during the pre-implantation period. Chromosome
analyses were made at the first cleavage mitosis, and on the 4th day following
transfer of the activated eggs at the pronuclear stage to the oviducts of pseudopregnant recipients. The early developmental potential of hypo- and hyperhaploid oocytes was compared with oocytes possessing the normal haploid
chromosome complement.
MATERIALS AND METHODS
(a) Activation of eggs. Eggs were isolated from the ampullar region of the
oviduct of 8- to 12-week-old (C57BL x CBA-T6T6)F1 and (C57BL x CBALAQFj female mice (hereafter referred to as FXT6 and FjLAC respectively)
at 25-26 h after the human chorionic gonadotrophin (HCG) injection for
superovulation. The activation technique was similar to that described elsewhere
(Kaufman, 1913 a, b; Kaufman & Surani, 1974), the only modification being
that all culture was carried out under light paraffin oil in 60 x 15 mm plastic
Petri dishes (Falcon plastics, No. 3002) instead of in embryological watchglasses. Five to six hours after activation the eggs were examined to determine
the overall activation frequency and types of parthenogenones induced.
Eggs which developed a single pronucleus following extrusion of the second
polar body were either retained in culture or isolated at this stage and transferred to the oviducts of pseudopregnant recipients. In the experiments where
activated eggs were allowed to remain in standard embryo culture medium
(Whittingham, 1971) for a further 6 h, embryos were transferred to fresh
medium containing 0-5-1 /^g/ml colchicine or colcemid and then examined by
air-drying (Tarkowski, 1966) 10-12 h later. By this time, nearly all the embryos
had entered the first cleavage mitosis. The preparations were stained with 3 %
Haploid and aneuploid parthenogenetic development
641
lactic acetic orcein or 4% Giemsa. The number of chromosomes present in
each metaphase group was recorded.
(b) Observations on the chromosome complement of oocytes at ovulation. F{T6
and FjLAC females were superovulated and their eggs isolated at approximately
14-16 h after HCG. The eggs were released directly into medium containing
hyaluronidase to remove adherent cumulus cells, and transferred after 5-10 min
to 1 % sodium citrate solution before air-drying (Tarkowski, 1966). A maximum
of three oocytes were carefully placed on each microslide.
(c) Oviduct transfer and the recovery of embryos from recipients on the 4th
day of pseudopregnancy. Eight- to twelve-week-old spontaneously ovulating
FjLAC and FXT6 females were examined by vaginal inspection (Champlin,
Dorr & Gates, 1973) at approximately 8 p.m. and those in oestrus were mated
to proven sterile vasectomized males. Females with vaginal plugs the following
morning were later used as recipients. The day on which a vaginal plug was
found was considered the first day of pseudopregnancy. All mice were kept
under similar lighting conditions, where the period of darkness lasted from
3 p.m. to 8 a.m.
Activated eggs were transferred to the right oviduct (Kaufman & Gardner,
1974), generally in batches of 10-12, between 3 and 5 p.m. on the afternoon of
the first day of pseudopregnancy. All the mice were anaesthetized with 0-02 ml/g
body weight of a freshly prepared 1:80 solution of avertin dissolved in 0-9 %
saline.
Recipients were killed on the 4th day of pseudopregnancy and both uterine
horns flushed with phosphate-buffered medium (Whittingham & Wales, 1969).
In ten females, the oviducts were also flushed with medium but no eggs were
recovered. All cleaving embryos more advanced than the 2-cell stage were
transferred to standard embryo-culture medium containing 0-5-1 ^g/ml
colchicine or colcemid, and incubated in this medium for 4-6 h. Embryos were
then examined by air-drying, and stained as described in section (a). The
number of blastomere nuclei present was recorded, as was the chromosome
number of the cells in mitosis.
RESULTS
(a) The chromosome complement of oocytes at ovulation and of embryos at the
first cleavage metaphase *
Analysis of diakinesis/metaphase I chromosomes from oocytes isolated at the
germinal vesicle stage and cultured for 4 h in standard embryo-culture medium,
has shown that all F{T6 oocytes had either a quadrivalent or a univalenttrivalent configuration (Fig. 1 A, B). No such chromosome configurations were
observed in the FiLAC oocytes. The F3T6 showed a much higher frequency of
aneuploid oocytes (37-8%) than the FXLAC oocytes (3-4%). The majority of
the aneuploid oocytes in the FjT6 had 19 or 21 chromosomes with about equal
frequency, and the one aneuploid out of 29 FXLAC oocytes examined had
1. Metaphase II (recently ovulated
oocytes)
2. First cleavage metaphase
3. Embryos recovered on the 4th day
1. Metaphase II (recently ovulated
oocytes)
2. First cleavage metaphase
3. Embryos recovered on the 4th day
Stage
81
59
28
59
50
15
12
1
1
2
110
74
29
61
57
46
20
74
19
7
16
14
—
12
21
Chromosome number
22
3-3
2-0$
3-4
26-4
20-5
37-8
Aneuploid
FXT6
FiLAC
F x hybrid
1284
623
Total no. of eggs
examined
35 (3-8)
20 (4-4)
Activated eggs (%):
immediate
cleavage
1 (0-1)
1 (0-2)
1 pronucleus
Nil 2PB*
31 (3-3)
8 (1-8)
2 pronuclei
* Second polar body.
t The percentage of the total eggs activated is given in parentheses.
863 (92-8)t
428 (93-7)
1 pronucleus
+ 2PB*
72-4
73-4
Overall activation
frequency (%)
Table 2. The pathways of development of parthenogenetic embryos at 5-6h after in vitro activation
* Embryos with 40 or 42 chromosomes are also included in these data.
t The T6 marker chromosome was present in the metaphases of 6 of these 7 embryos, and these embryos must therefore have originated
from the FXT6 recipients.
% The aneuploid embryos originating from the FXT6 recipients are not included in this calculation.
FXLAC
FXT6
Fjhybrid
Total no. of oocytes or
embryos examined
Table 1. The chromosome complement of oocytes at ovulation; and of parthenogenetic embryos
at the first cleaving metaphase and on the 4th day of development*
O
in
r
o
ffl
oo
Haploid and aneuploid parthenogenetic development
649
19 chromosomes (Table 1). There was also a higher incidence of aneuploids in
FXT6 (26-4%) compared to F^LAC (3-3%) eggs at metaphase of the first
cleavage mitosis. As at ovulation, the majority of aneuploids at the first cleavage
metaphase had 19 or 21 chromosomes in the FXT6, and the two aneuploids in
the FjLAC both had 19 chromosomes (Table 1).
About 93% of the FXT6 and FXLAC activated oocytes developed a single
pronucleus following extrusions of the second polar body (Table 2).
(b) The chromosome complement and cell number of embryos on the 4th day of
development
A total of 511 activated 1-cell F2T6 eggs were transferred to 44 (34 FXLAC
and 10 F]T6) recipients, and 412 cleaving embryos were recovered from the
uterine horns on the side of transfer on the 4th day of pseudopregnancy. Two
hundred and seventy-six embryos were more advanced than the 2-cell stage,
but 16 of these were lost during the air-drying procedure. One hundred and
thirty-six of the 260 embryos had cells in mitosis and unequivocal chromosome
counts could be made in 74 of these embryos. Thirteen of these 74 embryos had
21 chromosomes, one embryo had 42 chromosomes, and one 4-cell embryo had
a single metaphase with 19 chromosomes (Table 1). The T6M chromosome was
absent in the embryo with 19 chromosomes, but present in all of the embryos
with 21 chromosomes (Fig. 1C). One of the embryos with 21 chromosomes also
contained a large metacentric chromosome.
A total of 284 activated 1-cell FXLAC eggs was transferred to 24 FXT6
recipients, and 276 cleaving embryos were recovered from the uterine horns on
the side of transfer. One hundred and ninety-one embryos were more advanced
than the 2-cell stage, but 17 of these were lost during the air-drying procedure.
Seventy-eight of the 174 embryos had cells in mitosis and unequivocal chromosome counts could be made in 57 of these embryos. Seven of these 57 embryos
had 21 chromosomes. The T6M chromosome was absent in one of these
embryos with 21 chromosomes (Fig. ID). As the six embryos with 21 chromosomes in which the T6M chromosome was present were all isolated from FjT6
recipients, these could only have originated from activation of the recipients' ova.
Of the FjT6 embryos recovered on the 4th day 10-3 % were ' haploid/diploid'
mosaics. Thirteen embryos were 20/40 mosaics and one embryo was a 21/42
mosaic (Table 3). These mosaics have been classified under the groups with
20 or 21 chromosomes in the data in Table 1, as have the 'diploid' embryos with
40 or 42 chromosomes. The classification was based on chromosome counts of
a mean of between three and four cells in mitosis per embryo. Of the FjT6
embryos 8-1% had 40 and 1-4% had 42 chromosomes. All of the FjLAC
'diploids' had 40 chromosomes.
The mean cell number of eleven F2T6 embryos with 21 chromosomes recovered from the transfer side and a further five similar embryos recovered
from the control series was 11-7, while the mean cell number of 49 ¥{T6
650
M. H. KAUFMAN AND L. SACHS
A
t
5//m
D
5/mi
Haploid and aneuploidparthenogenetic development
651
embryos with 20 chromosomes was 12-0. The mean cell number of all the
embryos which were more advanced than the 2-cell stage was similar in the
FjT6 and FjLAC series (Table 3).
(c) The chromosome complement and cell number of eggs and embryos recovered
from the control uterine horns of recipients on the 4th day ofpseudopregnancy
As avertin anaesthesia may induce parthogenetic development (Kaufman,
1975), the control uterine horns also were studied in recipients on the 4th day
of pseudopregnancy. A total of 36 and 47 cleaving embryos was recovered
from the control uterine horns of 29 FXT6 and 25 FXLAC recipients, respectively.
Thirty of the F3T6 and 35 of the FXLAC embryos were more advanced than
the 2-cell stage. The mean number of cleaving embryos recovered from the
FXT6 and FXLAC control uterine horns was therefore 1-2 and 1-9 per horn
respectively.
Fifteen of the FjT6 and 24 of the F2LAC embryos had cells in mitosis. All
five of the FjT6 embryos in which unequivocal counts could be made had 20
chromosomes. Thirteen of the F3LAC embryos in which unequivocal counts
could be made had 20 chromosomes, while two embryos had an aneuploid
chromosome complement. One of these aneuploid embryos had 21 chromosomes (the T6M was not present), while the other had 41 chromosomes, one of
which was a large metacentric chromosome (Fig. 1E). The mean cell number
of the F]T6 and F a LAC embryos which were more advanced than the 2-cell
stage was 10-2 and 13-4, respectively.
In 9 out of 34 F/T6 recipients and 8 out of 34 F3LAC recipients in which
more cleaving embryos were recovered from the uterine horns than activated
1-cell eggs were transferred, the mean number of extra cleaving embryos was
1*3 and 1-5 per horn respectively.
FIGURE 1
Air-dried chromosome preparations. A-D stained with Giemsa, E stained with
lactic acetic orcein. The bar on each photograph represents 5 /tm.
A. FXT6 (T6 translocation heterozygote) oocyte at diakinesis-metaphase I, with
18 bivalents and a chain quadrivalent (arrowed).
B. F{T6 oocyte at diakinesis-metaphase I, with 18 bivalents, a univalent (the T6
marker chromosome, T6M, short arrow), and a trivalent (long arrow).
C. Metaphase plate with 21 chromosomes including the T6M (arrowed), from an
18-cell FXT6 parthenogenetic embryo.
D. Metaphase plate with 21 chromosomes from a 15-cell FXLAC parthenogenetic
embryo.
E. Metaphase plate with 41 chromosomes including a large metacentric chromosome (arrowed), from a 5-cell FjLAC parthenogenetic embryo.
'Haploid'*
106
66
260
174
396
259
3-4
93
59
2
136
85
92
69
5-8
60
37
9-16
Cell number
14
5
' Haploid/diploid '*
mosaics
8
7
17-32
16
7
7
2
33-64
'Diploid'*
8-7
7-8
Mean cell number
of embryos with
more than 2 cells
136
78
Total no. of embryos
with cells in
mitoisisf
* 'Haploid' includes 19-21 chromosomes, and 'diploid' includes 40-42 chromosomes.
t These embryos were scored as ' haploid', ' haploid/diploid' mosaics or ' diploids' without necessarily making an unequivocal count of
the chromosome number.
FXT6
F t LAC
Total no. of embryos
examined
07) Cell number of embryos recovered on the 4th day of development
F x hybrid
FXT6
F^AC
hybrid
Total no. of embryos
examined
Chromosome numbers
(/) Chromosome complement of embryos which were more advanced than the 2-cell stage
Table 3. The chromosome complement and cell number of parthenogenetic embryos recovered
on the 4th day of development
O
E
oo
ON.
Haploid and aneuploid parthenogenetic development
653
DISCUSSION
We have compared the early developmental potential of haploid and aneuploid oocytes isolated from genetically similar normal and translocationbearing mice. Oocytes, activated eggs and embryos from the translocationbearing F3T6 mice had a much higher frequency of aneuploidy than those from
the non-translocation-bearing FjLAC mice. Eggs with 20 and 21 chromosomes
had a similar development, at least up to the morula stage. The mean cell
number of the embryos with 20 and 21 chromosomes was similar, while the
proportion of 21-chromosome embryos was about that expected from the
incidence of oocytes ovulated with 21 chromosomes. The presence of this
additional chromosome, therefore, did not hinder the early haploid embryonic
development. Embryos with 20 chromosomes which have a balanced genetic
constitution can contain either the normal chromosomes 14 and 15, or the
small (T6M) and large translocation products. Embryos with 21 chromosomes
must contain three out of four of these normal and translocation products,
following non-disjunction at meiosis I. All the embryos with 21 chromosomes
from the FjT6 series contained the T6M, in contrast to the frequency of 50 %
expected if the distribution were random. The two embryos with 21 and 41
chromosomes (one of the 41 chromosomes was a large metacentric), which did
not include a T6M chromosome, both came from the F a LAC series, and may
have been disomic for chromosomes other than numbers 14 or 15.
There were many oocytes and activated eggs with 19 chromosomes in the
FjT6. The observation of only a single 4-cell embryo with 19 chromosomes out
of 129 analysable embryos recovered from both the transfer and control sides
in the F3T6 and FXLAC series, suggests that this condition is almost certainly
inviable very early in development. Most embryos with 19 chromosomes
probably fail to develop beyond the 2-cell stage. Contraction of the chromosomes as a result of the colchicine or colcemid treatment prevented identification
of the T6M chromosome in the first cleavage metaphase, and the single 4-cell
embryo with 19 chromosomes did not contain a T6M chromosome. Fewer
cleaving embryos were recovered in the F3T6 than the FXLAC series (80-6%
and 97-2%, respectively, of the embryos transferred). When the number of
embryos recovered from the control uterine horns is taken into account, the
proportion of transferred embryos recovered was probably nearer 66 % in the
FjT6 and 87 % in the FjLAC series, respectively. The reduction of the 19chromosome population, from approximately one-fifth of the activated eggs
on day 1 to only one embryo recovered on the 4th day in the FXT6 series,
could account for the reduced recovery rate of cleaving embryos in the FiT6
series. Haploid FjT6 parthenogenetic embryos may also die very early in
development because they contain an unbalanced genome following chiasma
formation at meiosis I (Searle et al. 1971).
Fertilization of oocytes with 21 chromosomes can lead to the production of
654
M. H. KAUFMAN AND L. SACHS
viable offspring with 41 chromosomes which are T6M trisomics. In contrast to
the high rate of early development of the parthenogenetic embryos with 21
chromosomes, the 9-day-old embryonic (Evans & Meredith, reported as
personal communication in Cattanach, 1967), and post-natal incidence of
mice with 41 chromosomes produced after fertilization was 2-7% (Cattanach,
1967; Eicher & Green, 1972). These results suggest that most embryos with the
additional T6M chromosome die later.
All the embryos transferred, and most of those originating from activation
of the recipients' own ova, were initially haploid. Thus the high incidence of
embryos with 20 or 21 chromosomes recovered on the 4th day, rather than
haploid/diploid mosaics or diploid embryos, suggests that the process of cell
division is normal in most of the haploids.
Data from the FXLAC series confirm the observation of Graham & Deussen
(1974), that culture of oocytes in medium of normal osmolarity directly after
activation in vitro does not induce non-disjunction at meiosis II. The very low
incidence of aneuploidy observed in recently ovulated oocytes from 8- to 12week-old FXLAC mice also supports previous observations that the occurrence
of non-disjunction at meiosis I is rare in oocytes from young non-translocationbearing mice (Rohrborn, 1972, and following discussion; Uchida & Lee, 1974).
However, even in non-translocation-bearing animals there can be more aneuploidy as a result of non-disjunction at meiosis I in old females (Henderson &
Edwards, 1968; Gosden, 1973; Yamamoto, Shimada, Endo & Watanabe, 1973).
The further study of aneuploid embryos should be able to determine which
other genetic deficiencies or duplications influence embryonic viability. The
present experimental approach should therefore prove useful in further elucidating the genetic basis of normal and abnormal embryonic development.
M.H.K. is a recipient of an MRC Travelling Fellowship.
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