/. Embryol. exp. Morph. Vol. 71, pp. 139-154,1982
Printed in Great Britain © Company of Biologists Limited 1982
\ 39
The chromosome complement of
single-pronuclear haploid mouse embryos following
activation by ethanol treatment
By M. H. KAUFMAN 1
From the Department of Anatomy, University of Cambridge
SUMMARY
A technique in which mouse eggs are stimulated to develop parthenogenetically following
their brief incubation in 7 % ethanol in PBS is described. Very high rates of activation were
achieved, and a detailed analysis presented of the class of parthenogenone which develops a
single haploid pronucleus following second polar body extrusion.
As preliminary studies on presumptive haploid morulae indicated that a proportion of the
metaphase spreads examined had an aneuploid chromosome constitution, the incidence of
aneuploidy at the first cleavage mitosis was investigated. In the control groups the level of
aneuploidy was about 1 %, whereas in the ethanol-treated series the incidence ranged from
13-6-18-8%. Additional pre-treatment of ethanol-activated oocytes in low osmolar medium
raised the incidence of aneuploidy to 28-3%. Metaphase groups with 18, 19, 21 and 22
chromosomes present were observed in addition to groups with a normal complement of 20
chromosomes. The possible mode of action of ethanol in inducing parthenogenetic activation
of mouse oocytes, and a high incidence of aneuploidy, is discussed in relation to previous
knowledge of the action of this agent. Preliminary studies using G-banding indicate that the
aneuploidy observed appears to arise as a result of non-disjunction which may involve any of
the chromosomes of the complement.
INTRODUCTION
The technique in which mouse eggs may be stimulated to develop parthenogenetically by incubating them for a short time in a 7 % solution of ethanol in
phosphate-buffered saline (7% ethanol in PBS) was first described by
Cuthbertson (personal communication). This technique originated from the
observations of Dyban & Khozhai (1980) who found that an intraperitoneal
injection of ethanol into mice about 7 h after ovulation induced activation of
the oocytes. The majority of eggs activated by the in vitro technique, when eggs
are stimulated approximately 17 h after the HCG injection for superovulation,
develop a single pronucleus following second polar body extrusion (1 pron +
2PB class, see Kaufman, 1981 a).
Optimum activation and culture conditions were established, so that a high
1
Author's address: Department of Anatomy, University of Cambridge, Downing Street,
Cambridge CB2 3DY.
140
M. H. KAUFMAN
proportion of the single pronuclear haploids subsequently developed to the
blastocyst stage. However, analysis of the chromosome constitution of embryos
at the early morula stage, initially carried out to confirm the haploid status of
these individuals, revealed that a proportion of the metaphase plates in both of
the strains examined ((C57BL x C B A ^ and 129/SvE) had an aneuploid
chromosome constitution. Activated embryos at metaphase of the first cleavage
mitosis were then examined, in order to establish the incidence of aneuploidy
shortly after activation. Attempts to investigate whether the aneuploidy arose
by non-disjunction or by chromosome aberrations or both were made by Gbanding, and these results are reported here.
The possible significance of the present findings regarding the exposure of
mouse oocytes during the second meiotic division to ethanol, and their relationship to earlier observations on the potential teratogenicity of this agent and its
principal breakdown product (see Kaufman, 1981/)) are discussed in detail.
MATERIALS AND METHODS
Eight- to 12-week-old (C57BL x CBA)Fj hybrid female mice were superovulated with 5 i.u. PMSG followed 48 h later by 5 i.u. HCG. In the majority of the
experiments described here, females were autopsied at 17 h after the HCG
injection. The oviducts were then removed and the individual cumulus masses
containing the ovulated oocytes released into PBS. Cumulus masses from four to
six females were pooled together, treated as a single group, and all of the
following procedures were carried out at room temperature. In the experimental
series, groups of cumulus masses were transferred via a Pasteur pipette to a
watchglass containing about 1 ml of a freshly prepared 7 % solution of Analar
quality ethanol in PBS, and retained in this solution for either 1 min, 3 min,
4 | min or 1\ min. The cumulus masses were then washed through three changes
of ethanol-free PBS and finally through two changes of embryo culture medium
(Whittingham, 1971). Individual cumulus masses were then transferred to
separate drops of culture medium under paraffin oil and incubated for 4-5 h at
37 °C in an atmosphere of 5 % CO 2 in air. At the end of this time, the adherent
cumulus cells were removed with hyaluronidase (Kaufman, 1978) and the overall
activation frequency determined, and the various classes of parthenogenone
induced separated into different groups. Depending on whether a second polar
body had been extruded or not, and the overall size of the latter - whether equal
in volume or considerably less than the volume of the oocyte, and on the number
of pronuclei formed, four classes of parthenogenone could be determined at this
stage, namely (a) oocytes which contained a single (haploid) pronucleus, having
previously extruded a second polar body, (b) oocytes which contained two
(haploid) pronuclei in the absence of second polar body extrusion, (c) oocytes
which underwent 'immediate cleavage' in which two equal-sized blastomeres
had formed, each containing a single (haploid) pronucleus, one of the blasto-
141
Ethanol-induced aneupJoidy
lOOi
• 1 ponucleus with 2nd polar body
(142)
M 2 pronuclei without 2nd polar body
1 pronucleus without 2nd polar b o d y n 2 6 )
(107)
Immediate cleavage
80-
Z
100
80
60
(69)
60
40
40
20
20
>
Time (min)
Fig. 1. The pathways of development of parthenogenetic eggs and overall incidence
of activation following their incubation in 7% ethanol in PBS for 1, 3, A\ and 1\
min. The eggs were isolated from (C57BL x CBA)Fi hybrid mice at 17 h after
HCG and the analysis was carried out at about 5 h after activation.
meres representing the second polar body, and (d) oocytes in which a single
(diploid) pronucleus developed in the absence of second polar body extrusion.
Apart from briefly considering how the proportionate incidence of these various
classes varied with the different treatments, only the haploid oocytes constituting
group (a) will be considered further.
At about 10 h after activation the oocytes in group (a) were transferred to
fresh medium containing 1 /^g/ml colcemid, and returned to the incubator.
After a further 10-12 h, the oocytes were isolated and their chromosome
constitution determined by the air-drying technique described by Tarkowski
(1966). All preparations were stained with Giemsa. Without exception, the
oocytes were inhibited from developing beyond metaphase of the first cleavage
mitosis. The total number of chromosomes present in individual metaphase
spreads was determined using the oil-immersion objective of a Leitz photomicroscope. If there was any doubt regarding the number of chromosomes
present due to the overlapping of chromosomes, the group was excluded from
the study. All of the aneuploid groups which contained either more or less than
the normal haploid complement of 20 chromosomes were photographed.
In addition to the four experimental groups already described, the chromosome constitution of oocytes from three control groups and a further experimental group were also analysed. The first control group consisted of oocytes
(1) Control
(2) Control
(3) Control (hyaluronidase) activation
(4) 1 min exposure
(5) 3 min exposure
(6) 4£ min exposure
(7) 7 | min exposure
(8) 4£ min exposure then 2 h in low osmolar
medium
Group
17
20
20
17
17
17
17
17
Activation
after
HCG(h)
+
+
+
+
+
48
81
85
52
60
10
42
37
_
— 10 — —
— — 42 — —
—
1 36 — —
1
3 39 4 1
1
7 70 3 —
4
7 69 5 —
1
7 44 - —
1
6 43 8 2
0
0
2-7
18-8
13-6
18-8
15-4
28-3
71-4
79-2
71-2
81-4
77-9
82-5
78-8
84-5
Total no. of Chromosome
metaphase
number
Percentage of
7% ethanol spreads ,r
scorable
in PBS
analysed 18 19 20 21 22 Aneuploid (%) preparations
Table 1. Chromosome complement of single-pronuclear eggs analysed at metaphase of the first cleavage division
c
s
z
M-l
Ethanol-induced aneuploidy
143
isolated, as in the experimental series, at 17 h after the HCG injection. In this
group the cumulus masses were retained in ethanol-free PBS for 4-| min, then
washed through three rinses of ethanol-free PBS etc., exactly as described
earlier. However, as the activation frequency in this group was rather low and
the number of haploid oocytes available for analysis correspondingly small, two
additional control groups were established.
In the second control group, the only factor which was varied from the first
control group was the time of isolation of oocytes. In this group, the oocytes
were isolated at 20 h after the HCG injection, as previous studies (Kaufman,
1973, 1978) had shown that increasing the post-ovulatory age of the oocyte at
the time of activation could markedly increase the activation frequency. In all
other respects this group was treated in exactly the same way as the first control
group. Oocytes in the third control group were activated by incubation for 10
min in medium containing hyaluronidase, according to the technique described
by Kaufman (1973).
An additional experimental group was examined to investigate the effect of
incubating oocytes previously activated by exposure for 4 | min to 7 % ethanol
for 2 h in embryo culture medium diluted with distilled water in the ratio of
three volumes of medium to two volumes of distilled water. The rationale for
this group was that it had previously been established that this form of treatment
could influence the activity of the second meiotic spindle apparatus (Graham,
1971, 1972; Kaufman & Surani, 1974), and might also increase the incidence of
non-disjunction.
All of the preparations were examined without reference to their source, and
the chromosome complement of all aneuploid groups confirmed by an independent observer.
In a preliminary study, (C57BL x CBA)Fi and 129/SvE oocytes isolated at
17 h after the HCG injection were activated by incubating them for 4£ min in
7 % ethanol in PBS, and the cumulus masses then cultured for 4-5 h as described
earlier. The activated oocytes in group (a) were then segregated out and transferred shortly afterwards (at about 15.00-16.00 h) to the oviducts of recipients
on the first day of pseudopregnancy (i.e., on the afternoon of the day on which
a vaginal plug had earlier been observed, following mating of the female with a
vasectomized male).
The recipients were autopsied at about midday on the 4th day of pseudopregnancy, and the recovered embryos transferred to culture medium containing
1 /*g/ml colcemid. The majority of the embryos were at the morula stage. After
an incubation period of about 3 h the embryos were examined by the air-drying
technique (Tarkowski, 1966). The preparations were then stained with Giemsa,
and examined under the oil-immersion objective of a Leitz photomicroscope.
The total cell number of each embryo was determined, as well as its chromosome
constitution and ploidy. However, only the chromosome constitution will be
considered here, as their ploidy and pre- and early post-implantation development potential will be discussed in detail elsewhere (Kaufman & Handyside, in
preparation).
144
(a)
M. H. KAUFMAN
(b)
I
(c)
I
(e)
(/)
(/>)
Fig. 2. Representative air-dried chromosome spreads of the first cleavage metaphase of single-pronuclear haploid parthenogenetic eggs. All oocytes were incubated
for about 12 h in medium containing 1 /tg/ml colcemid, Metaphase spreads with
18 (a), 19 (b), 20 (c, d), 21 (e,f) and 22 (g, h) chromosomes present. The condensed
round object in b, d, e and g is the nucleus of the second polar body. The preparations were stained with Giemsa.
Ethanol-induced aneuploidy
145
G-banding analysis of first cleavage chromosomes
Oocytes for this analysis were either incubated in medium containing Colcemid,
or in the absence of this agent. In both groups, air-dried preparations were made
as soon as the pronuclear outline disappeared, as this indicated their entry into
the first cleavage mitosis. This event usually occurred within 15-18 h after
activation.
G-banding analysis of the preparations was obtained by a modification of the
A.S.G. technique described originally for rat chromosomes by Gallimore &
Richardson (1973). Banded spreads were photographed and karyograms
prepared according to the nomenclature suggested by Nesbitt & Francke
(1973).
RESULTS
(1) Observations on the activation rate and proportionate incidence of the various
classes of parthenogenone induced under various control and experimental
conditions
Observations on the various classes of parthenogenone induced when oocytes
isolated 17 h after HCG injection were stimulated by exposure to a 7% solution
of ethanol in PBS for 1 min, 3 min, 4 | min and 1\ min are presented in diagrammatic form in Fig. 1. In the control group exposed to ethanol-free PBS for A\
min but otherwise treated in exactly the same way as these experimental groups,
the activation rate was 14-0%, and all 15 activated eggs observed were of the
single-pronuclear haploid type (group a). Several points of interest emerge.
First, that under control conditions the activation rate is extremely low, but it is
clear that the rate increases in relation to the duration of exposure to the 7 %
ethanol solution, with very high rates achieved after 3 min, 4£ min and 1\ min
exposure. Secondly, that the principal class of parthenogenone induced under
these conditions is haploid, and contains a single pronucleus which develops
shortly after the second polar body has been extruded. In only the 4^ min and
more particularly in the 1\ min groups were reasonable numbers of other classes
of parthenogenone observed (see Fig. 1).
Because of the diversity of the other groups, these will be considered individually. It is of interest that the results obtained in the two additional control
groups, in which oocytes were stimulated at 20 h after the HCG injection, were
almost identical. Thus in the second control group, in which oocytes were
incubated for 4 | min in ethanol-free PBS the activation rate was 45-2%,
whereas in the third control group, in which oocytes were activated in medium
containing hyaluronidase, the activation rate was 45-3%. In the second
control group 54 single-pronuclear haploid eggs and 2 two-pronuclear diploid
eggs were observed, whereas in the third control group 52 single-pronuclear
haploid eggs and 6 two-pronuclear eggs were observed.
The results of the additional experimental study in which oocytes isolated 17 h
146
M. H. KAUFMAN
•
0
Ethanol-induced aneuploidy
147
after HCG were released into 7 % ethanol solution for 4 | min, then incubated in
medium diluted in the ratio of 3:2 with distilled water was also of considerable
interest. The activation rate observed in this group was 97-1 %, and out of a
total of 102 activated eggs, 89 were of the single-pronuclear haploid class, 9
were of the two-pronuclear diploid class and 4 eggs were observed which
underwent immediate cleavage. This result was unexpected, in that there
appeared to be little or no obvious effect of the incubation in the low osmolar
medium. Previous studies in which activated eggs had been incubated in low
osmolar medium directly after activation had usually resulted in a high incidence
of eggs in which suppression of second polar body extrusion occurred, with the
development of two-pronuclear diploid eggs. A moderate incidence of eggs which
underwent immediate cleavage were also commonly observed (see Kaufman,
1978).
(2) Observations on the chromosome complement of single-pronuclear haploid eggs
at metaphase of the first cleavage division
A summary of the findings in relation to the three control groups and five
experimental groups is presented in Table 1. It is of interest to note that the
incidence of scorable preparations, i.e. metaphase plates in which unequivocal
chromosome counts could be made, varied between 71-2 and 84-5%. These
figures would no doubt have been higher had groups with a minimal degree of
chromosome overlapping been included. However, as indicated earlier, where
there was any possible doubt about the number of chromosomes present, no
attempt was made to score these groups.
In none of the control groups were metaphases observed with more than the
normal haploid complement of 20 chromosomes. Indeed, in only one control
metaphase plate was a group with only 19 chromosomes present recorded.
Of the first four experimental groups in which the oocytes were incubated in
7 % ethanol in PBS for between 1 min and 1\ min, there seemed little difference
in the overall incidence of aneuploid chromosome preparations observed.
The incidence of preparations with less than 20 chromosomes was almost
exactly balanced by the number of preparations observed in which there were
Fig. 3. Representative air-dried chromosome spreads of single-pronuclear derived
morulae. (a) Metaphase group with 21 chromosomes present from a 7-cell Ft
hybrid morula. This embryo contained 5 metaphase spreads each of which
contained 21 chromosomes, (b) Metaphase group with 21 chromosomes present from
a 19-cell 129/SvE morula. This embryo contained 7 metaphase spreads each of
which was haploid. In most of these spreads 21 chromosomes could be distinguished,
(c) Metaphase group from a 7-cell Fj. hybrid haploid-diploid mosaic embryo. Six
metaphase spreads were present, one of which appeared to contain a giant chromosome (arrowed), {d) Metaphase group from an 18-cell Fx hybrid morula which
contained 6 haploid metaphases. The spread to the left contains 20 normal chromosomes while the partial spread on the right appears to contain a metacentric
chromosome (arrowed).
148
M. H. KAUFMAN
(a)
(b)
2
11
16
12
17
4
3
13
18
5
9
10
14
15
19
149
Ethanol-induced aneuploidy
(0
(d)
i
(
10
7
I
11
12
16
17
13
18
14
19
Fig. 4. Two G-banded aneuploid first cleavage chromosome spreads containing
19 chromosomes, (a) Metaphase spread in which chromosome number 4 is absent.
(b) Karyogram of this spread, (c) Metaphase spread in which chromosome number 9
is absent, id) Karyogram of this spread.
150
M. H. KAUFMAN
more than 20 chromosomes present. However, in the fifth experimental group in
which the activated oocytes were incubated for 2 h in low osmolar medium, the
incidence of aneuploidy was markedly increased from about 14-19% as
observed in the other experimental groups to 28-3%. As in the other experimental groups, an approximately similar incidence of metaphase preparations
with less than 20 and more than 20 chromosomes present was observed.
Representative first cleavage metaphase spreads with complements of 18, 19,
20, 21 and 22 chromosomes are illustrated in Fig. 2. As a result of the extended
colcemid treatment the chromosomes were quite condensed, and would probably
not have been suitable for' banding' analysis, even if they had been appropriately
pretreated prior to staining. However, their condensed morphology undoubtedly facilitated the present analysis in which only the total number of chromosomes present was of immediate interest. Neither metacentrics nor other
morphologically abnormal chromosomes were seen at this stage of development.
(3) Observations on the chromosome complement of single-pronuclear haploid
embryos at the morula stage of development
In the preliminary study, air-dried preparationsof 44 morulae from (C57BL x
CBA)F1 and 17 morulae from 129/SvE strain mice in which metaphase plates
were present were analysed. Of the ¥x preparations in which unequivocal
chromosome counts could be made, two embryos contained several metaphase
plates in which groups of 21 chromosomes were present. In two additional
embryos morphologically abnormal chromosomes were observed. In one case a
'giant' chromosome was present, and in another an apparent metacentric
chromosome was present in a single group but not in another spread in the same
embryo. In yet another embryo, only a single metaphase spread was present,
but this contained 25 chromosomes. Of the 129/SvE embryos examined,
two morulae contained several metaphase plates in which groups of 21 chromosomes were present. In addition, two morulae each contained a single metaphase
spread with only 19 chromosomes present, and a third morula, which also had
only a single metaphase spread present, apparently contained a complement of
24 chromosomes. The mean number of cells present in the F x group was 12-8,
and that in the 129/SvE group was 13-6. A selection of the metaphase spreads
containing abnormal chromosome complements is illustrated in Fig. 3.
In a proportion of the morulae, due to problems with overlapping chromosomes, only the ploidy could be determined. It is possible therefore that the true
incidence of aneuploidy at this stage of development could be considerably
higher than reported here.
(4) The results ofG-banding analyses of the first cleavage metaphase chromosomes
Attempts to analyse several hundred first cleavage metaphase spreads by Gbanding were only moderately successful, as it was frequently found that
preparations which banded well often had one or more groups of overlapping
Ethanol-induced aneuploidy
151
chromosomes, which made any detailed analysis of the complement impossible.
The chromosomes in other preparations which failed to band were often found
to be severely contracted, despite only a brief period of incubation in Colcemid.
In 10 groups in which 20 chromosomes were present, all of the autosomes and
a single X chromosome were recognized. In 3 additional groups, only 19 chromosomes were present. The chromosomes which were found to be absent in these
metaphases were autosome numbers 4, 9 and 16, respectively. The first two of
these banded preparations and their karyotypes are illustrated in Fig. 4.
DISCUSSION
The present study has clearly demonstrated that exposure of mouse oocytes to
a dilute solution of ethanol is an extremely effective activating stimulus. Maximum
rates of activation were achieved after exposure to a 7 % solution of ethanol in
PBS lasting about 3-4£ min. However, the most interesting finding to emerge
from the present study was the observation that ethanol induced chromosome
non-disjunction in almost 19% of the haploid oocytes exposed to this agent. It
was also apparent that the incidence of non-disjunction could be substantially
increased if oocytes previously activated with ethanol were incubated in low
osmolar medium during the time that the oocytes were completing the second
meiotic division.
Analyses of the chromosome complements of the first cleavage metaphase in
the three control studies presented here clearly demonstrate that in (C57BL x
CBA)Fi hybrid mice the incidence of non-disjunction is extremely low. This
observation merely confirms similar analyses (Kaufman & Sachs, 1975) in which
the chromosome complement of recently ovulated oocytes and first cleavage
metaphase groups from the same ¥x hybrid mice had been determined. In this
earlier study, one out of a total of 29 metaphase II groups contained 19 chromosomes, as did two out of 61 first cleavage metaphases analysed. As in the present
study, none of the control groups analysed contained more than 20 chromosomes. This observation is also in accord with the conclusions of Rohrborn
(1972) and Uchida & Lee (1974) that the occurrence of non-disjunction at
meiosis I is rare in oocytes from young non-translocation bearing mice. However, even in these stocks there appears to be a sharp increase in the incidence of
non-disjunction at meiosis I in aged females (Henderson & Edwards, 1968;
Gosden, 1973; Yamamoto, Shimada, Endo & Watanabe, 1973).
In the four experimental groups in which cumulus masses were exposed to
7% ethanol in PBS for 1, 3, 4 | and 1\ min, a relatively high incidence of nondisjunction was observed (13-6-18-8 %). In the first three of these groups
aneuploid preparations were observed which contained 18, 19, 21 and 22
chromosomes, whereas in the fourth group only preparations with 18 and 19
chromosomes were observed. Considering the relatively small number of
aneuploid preparations analysed in the present study, it is not clear whether the
152
M. H. KAUFMAN
increase in the incidence of hypohaploidy which appears' to be related to
increasing exposure to ethanol is a real effort or not. It is curious that 82% of
the aneuploid preparations contained either 19 or 21 chromosomes, and the
present findings give no clear indication whether one or several chromosomes
are particularly commonly involved in the non-disjunctional event or all of the
chromosomes are equally susceptible. The only certain way to distinguish
between these two possibilities obviously involves subjecting an additional
group of preparations obtained under similar conditions to those described here
to appropriate pretreatment, for example, using a technique similar to that
employed by Nesbitt & Donahue (1972), and subsequently analysing the
'banded' chromosomes obtained. In the preliminary G-banding study reported
here, in the three aneuploid metaphase spreads with 19 chromosomes present, a
different autosome was involved in each of these groups.
The fact that a dilute solution of ethanol is an efficient activating agent is not
altogether surprising. It may well be that the underlying mechanism may be
similar to that occurring in the case of exposure of mouse oocytes in vitro to local
anaesthetics and phenothiazine tranquillizers as demonstrated by Siracusa,
Whittingham, Codonesu & De Felici (1978), and in vivo to Avertin (Kaufman,
1975). Their ability to initiate activation may be related to their effect on several
cellular processes which are most probably inter-related, though the exact interrelationships remain unclear. These processes include displacement of Ca 2+
from membrane phospholipids (Nicolson, 1976), changes in intracellular pH
(Johnson, Epel & Paul, 1976), the disruption of the cellular cytoskeletal system
(Nicolson, 1976), changes in the electrical properties of the egg membrane
(Seeman, Chen, Chan-Wong & Staiman, 1974), and finally changes in membrane fluidity (Sheetz & Singer, 1974). A recent study (Ahuja, 1982) has suggested that similar mechanisms may be involved during the activation of hamster
oocytes by procaine and tetracaine.
The observation reported in the present study, that a high incidence of
aneuploidy may be induced when a population of recently activated oocytes are
incubated in a dilute solution of ethanol, may also be explicable in the light of
previous knowledge of the action of this and other related agents, for example,
on the cellular cytoskeletal system either directly or indirectly via other intracellular changes mentioned above. The ultrastructural studies on the teratogenic
effect of acetaldehyde (O'Shea & Kaufman, 1979, 1981), the primary metabolic
product of ethanol oxidation, for example, and xylocaine (O'Shea & Kaufman,
1980) on early-somite-stage mouse embryos clearly demonstrated that these
agents acted on both the microtubular and microfilamentous components of the
cytoskeletal system, though the exact mode of action of these treatments is
unclear (for recent review, see O'Shea, 1981).
Studies on the effect of ethanol administration to pregnant mice have also
clearly demonstrated the rapidity with which this agent reaches the foetal
compartment (Kaufman & Woollam, 1981) and its various teratogenic effects
Ethanol-induced aneuploidy
153
on embryonic and foetal development (Sandor & Amels, 1971; Kronick, 1976;
Brown, Goulding & Fabro, 1979; Randall & Taylor, 1979), though detailed
ultrastructural studies are lacking. It should uot be surprising therefore that this
agent may also interfere with chromosome segregation, as the relationship
between the spindle apparatus and the centromere is both complex and finely
balanced (Hughes, 1952).
This work was supported by grants from the Medical Research Council and the National
Fund for Research into Crippling Diseases. I thank Kamal Ahuja for helpful discussion and
Mrs Lesley Cooke for technical assistance. The G-banding and karyotyping studies were
carried out by Elizabeth Robertson, and her assistance in this aspect of the work is gratefully
acknowledged.
REFERENCES
K. K. (1982). In vitro inhibition of the block to polyspermy of hamster eggs by
tertiary amine local anaesthetics. /. Reprod. Fert. 65, 15-23.
BROWN, N. A., GOULDING, E. H. & FABRO, S. (1979). Ethanol embryotoxicity: direct effects
on mammalian embryos in vitro. Science, N. Y. 206, 573-575.
DYBAN, A. P. & KHOZHAI, L. I. (1980). Parthenogenetic development of ovulated mouse ova
under the influence of ethyl alcohol. Bull. exp. Biol. Med. 89, 528-530.
GALLIMORE, P. H. & RICHARDSON, C. R. (1973). An improved banding technique exemplified
in the karyotype analysis of two strains of rat. Chromosoma 41, 259-263.
GOSDEN, R. G. (1973). Chromosomal anomalies of preimplantation mouse embryos in
relation to maternal age. /. Reprod. Fert. 35, 351-354.
GRAHAM, C. F. (1971). Experimental early parthenogenesis in mammals. Adv. Biosci. 6,
87-97.
GRAHAM, C. F. (1972). Genetic manipulation of mouse embryos. Adv. Biosci. 8, 263-273.
HENDERSON, S. A. & EDWARDS, R. G. (1968). Chiasma frequency and maternal age in
mammals. Nature, Lond. 218,22-28.
HUGHES, A. (1952). The Mitotic Cycle. New York: Academic Press.
JOHNSON, J. D., EPEL, D. & PAUL, M. (1976). Intracellular pH and activation of sea urchin
eggs after fertilization. Nature, Lond. 262, 661-664.
KAUFMAN, M. H. (1973). Parthenogenesis in the mouse. Nature, Lond. 242, 475-476.
KAUFMAN, M. H. (1975). Parthenogenetic activation of mouse oocytes following avertin
anaesthesia. /. Embryol. exp. Morph. 33, 941-946.
KAUFMAN, M. H. (1978). The experimental production of mammalian parthenogenetic
embryos. In: Methods in Mammalian Reproduction (ed. J. C. Daniel, Jr.), pp. 21-47.
San Francisco: Freeman.
KAUFMAN, M. H. (1981a). Parthenogenesis: a system facilitating understanding of factors that
influence early mammalian development. In: Progress in Anatomy, vol. 1 (ed. R. J. Harrison
& R. L. Holmes), pp. 1-34. Cambridge University Press.
KAUFMAN, M. H. (19816). The role of embryology in teratological research, with particular
reference to the development of the neural tube and heart. /. Reprod. Fert. 62, 607-623.
KAUFMAN, M. H. & SACHS, L. (1975). The early development of haploid and aneuploid
parthenogenetic embryos. /. Embryol. exp. Morph. 34, 645-655.
KAUFMAN, M. H. & SURANI, M. A. H. (1974). The effect of osmolarity on mouse parthenogenesis. /. Embryol. exp. Morph. 31, 513-526.
KAUFMAN, M. H. & WOOLLAM, D. H. M. (1981). The passage to the foetus and liquor amnii
of ethanol administered orally to the pregnant mouse. Br. J. exp. Path. 62, 357-361.
KRONICK, J. B. (1976). Teratogenic effects of ethyl alcohol administered to pregnant mice.
Am. J. Obstet. Gynec. 124, 676-680.
NESBITT, M. N. & DONAHUE, R. P. (1972). Chromosome banding patterns in preimplantation
mouse embryos. Science, N. Y. 177, 805-806.
NESBITT, M. N. & FRANCKE, U. (1973). A system of nomenclature for band patterns of mouse
chromosomes. Chromosoma 41, 145-158.
AHUJA,
154
M. H. KAUFMAN
G. L. (1976). Transmembrane control of the receptors of normal tumor cells.
Biochim. biophys. Ada. 457, 57-108.
O'SHEA, S. (1981). The cytoskeleton in neurulation: role of cations. In: Progress in Anatomy,
vol. 1 (ed. R. J. Harrison & R. L. Holmes), pp. 35-60. Cambridge University Press.
O'SHEA, K. S. & KAUFMAN, M. H. (1979). The teratogenic effect of acetaldehyde: implications for the study of the fetal alcohol syndrome. J. Anat. 128,65-76.
O'SHEA, K. S. & KAUFMAN, M. H. (1980). Neural tube closure defects following in vitro
exposure of mouse embryos to xylocaine. /. exp. Zool. 214, 235-238.
O'SHEA, K. S. & KAUFMAN, M. H. (1981). Effect of acetaldehyde on the neuroepithelium of
early mouse embryos. J. Anat. 132, 107-118.
RANDALL, C. L. & TAYLOR, W. J. (1979). Prenatal ethanol exposure in mice: teratogenic
effects. Teratology 19, 305-312.
ROHRBORN, G. (1972). Frequencies of spontaneous non-disjunction in metaphase II oocytes
in mice. Humangenetik 16, 123-125.
SANDOR, S. & AMELS, D. (1971). The action of aethanol on the praenatal development of
albino rats. Rev. roum. d'Embryol. Cytol. Ser a"Embryol. 8,105-118.
SEEMAN, P., CHEN, S. S., CHAN-WONG, M. & STAIMAN, A. (1974). Calcium reversal of nerve
blockage by alcohols, anesthetics, tranquilizers and barbiturates. Can. J. Physiol. Pharmacol. 52, 526-534.
SHEETZ, M. P. & SINGER, S. J. (1974). Biological membranes as bilayer couples. A molecular
mechanism of drug-erythrocyte interactions. Proc. natn. Acad. Sci., U.S.A. 71, 4457-4461.
SIRACUSA, G., WHITTINGHAM, D. G., CODONESU, M. & DE FELICI, M. (1978). Local anesthetics and phenothiazine tranquilizers induce parthenogenetic activation of the mouse oocyte.
DevlBiol 65, 531-535.
TARKOWSKI, A. K. (1966). An air-drying method for chromosome preparations from mouse
eggs. Cytogenetics 5, 394-400.
UCHIDA, I A. & LEE, C. P. V. (1974). Radiation-induced non-disjunction in mouse oocytes.
Nature, Lond. 250, 601-602.
WHITTINGHAM, D. G. (1971). Culture of mouse ova. /. Reprod. Fert. Suppl. 14,7-21.
YAMAMOTO, M., SHIMADA, T., ENDO, A. & WATANABE, G.-I. (1973). Effects of low-dose X
irradiation on the chromosomal non-disjunction in aged mice. Nature New Biology 244,
206-208.
NICOLSON,
{Received 21 December 1981, revised 5 April 1982)