/. Embryol. exp. Morph. Vol. 37, pp. 65-77, 1977
Printed in Great Britain
65
Effects of extremely low osmolarity on
fertilized mouse eggs
By JOLANTA OPAS
From the Department of Embryology, Institute of Zoology,
University of Warsaw
SUMMARY
When fertilized one-cell eggs are subjected to distilled water treatment for 2-6 min,
cytoplasm bulges through the sperm-slit in the zona pellucida and forms a cytoplasmic
fragment (CF). CFs were observed in 86-5 % of eggs; in 20-9 % of cases CFs contained
a pronucleus (or pronuclei). In 53-4 % of eggs permanent incorporation of the second polar
body (2 P.B.) into the egg cytoplasm occurred.
These phenomena occurring in different combinations produced 6-2 % of haploid eggs,
10-3 % of diploid eggs with a pronucleus replaced by 2 P.B. nucleus, and 43-1 % of triploid
eggs. 4-4 % of eggs were enucleated. The remaining group comprised diploid eggs which were
either not affected by the treatment (6-4 %) or lost a certain amount of cytoplasm by
formation of an anucleate CF (29-6 %). The frequencies of the types of reaction were related
to the post-fertilization stage of eggs.
All eggs except the enucleated ones were able to develop to the stage of morula or blastocyst. Triploids developed until the 12th day of pregnancy and diploids that had lost up to 15 %
of the cytoplasm developed to term. There was a twofold reduction in the percentage of
preimplantation development when treated eggs originated from induced rather than
spontaneous ovulation.
INTRODUCTION
Low osmolarity of culture media can produce cytological changes (suppression of second polar body formation, immediate cleavage) in parthenogenetically activated mouse eggs (Graham, 1971; Graham & Deussen, 1974),
as well as in fertilized ones (Niemierko, personal communication, but see also
Discussion, p. 75), which in turn have genetic consequences. Unfortunately, if
moderately hypotonic media are to be effective, prolonged treatment is necessary,
which simultaneously induce egg lysis (Graham & Deussen, 1974). In an
attempt to avoid these adverse effects a method has been developed to induce
cytological changes in fertilized mouse eggs by extremely low osmolarity
(distilled water) of very short duration. Escape of anucleate or pronucleate
cytoplasmic fragments and incorporation of the second polar body into the
cytoplasm occur under these conditions, producing various cytological and
karyological effects which are of interest from embryological and genetical
points of view.
1
Author's address: Department of Embryology, Institute of Zoology, University of
Warsaw, 00-927 Warszawa, Poland.
66
JOLANTA OPAS
MATERIALS AND METHODS
F x (C57BLxCBA/H) females, 4-6 weeks old, kept under a 16 h light/8 h
dark cycle centred on midnight, were used throughout this experiment. Females
were caged with A or CBA-T6T6 males in the afternoon and checked for
vaginal plugs next morning; the eggs were recovered between 0900 h and 1200 h.
Alternatively females were induced to ovulate (5 i.u. of PMSG and HCG each,
given 40-48 h apart); at the time of the second injection they were placed with
CBA-T6T6 males. Those that had mated were killed 18-5-20 h post-HCG and
cumuli oophori were removed in Ringer's solution containing 300 i.u./ml of
hyaluronidase. The eggs were then collected in Ringer's solution with 0-1 % of
plasma bovine albumin.
The eggs were individually placed in drops of distilled water under paraffin
oil at room temperature and kept under constant observation for 2-6 min. In
order to stop the reaction at the desired stage a small amount of medium was
added to the drop of water and both the egg and its cytoplasmic fragment (if
present) were transferred to a drop of culture medium in the same Petri dish.
All manipulations were performed under a dissecting microscope.
Eggs were cultured in Whitten's medium (Whitten, 1971) in siliconized glass
Petri dishes, under paraffin oil, in an atmosphere of 5 % CO2 in air for 3-5 h.
After this period the effects of water shock were determined under an inverted
microscope. In some experiments diameters of treated eggs and their CFs were
measured with a micrometer eyepiece and the percentage of egg volume
reduction was calculated.
Some eggs were then fixed and mounted as permanent preparations
(Tarkowski & Wroblewska, 1967); others were transplanted into the oviduct of
immature or pseudopregnant recipients on the first day of pseudopregnancy
(Tarkowski, 1959). Eggs of each type were usually transferred to separate
recipients. The transplanted embryos were recovered on the 4th or 5th day of
development or after implantation. Chromosome preparations were made of
morulae and blastocysts (Tarkowski, 1966) and of post-implantation embryos
by the technique of Evans, Burtenshaw & Ford (1972).
RESULTS
Reaction of eggs to water shock
During the first minute of treatment intense swelling, rupture of the first
polar body (if still present) and compression of the second polar body (2 P. B.)
to the egg take place in all treated eggs. Between the 1st and 3rd minute of
treatment, cytoplasm bursts out from many eggs and forms a round cytoplasmic
projection (later referred to as cytoplasmic fragment-CF) connected with the
egg by a very thin cytoplasmic stalk. During further treatment CFs enlarge
as more cytoplasm flows out of the eggs. The process may stop spontaneously
Effects of low osmolarity on mouse zygotes
67
CF
Present:86-5 "•'
Absent : 13-5
No PR in CF : 65-6 %
1:6-4'!;
PR in CF : 20-9 °'o
II: 29-6 " 0
o
Present
46-6 "„
2 P.B.
VII
Absent
53-4 "i
VI : 3 6 0 ° ;
V: 10-3 %
O
©
Fig. 1. Types of eggs produced by water shock and their frequencies. CF, cytoplasmic fragment; 2 P.B., second polar body: PR, pronucleus. * This group was not
homogeneous; for explanation, see text.
or continue until the egg ruptures. In the former case eggs and CFs were
transferred to the culture medium just after the CF stopped enlarging (2-4 min);
in the latter case the moment of transferring eggs was selected so as to obtain
the biggest CF and avoid egg rupture (4-6 min). Between the 3rd and 5th minute
of water treatment some 'resistant' eggs start to produce a CF. These CFs are
usually small, however, and rarely enlarge to exceed their initial size. All eggs
with or without CFs were removed from distilled water and placed in medium
within 5-6 min, since longer treatment usually caused lysis.
The cytoplasm of eggs and CFs looks normal after 5-10 min in culture
medium. Some eggs re-extrude 2 P.B. during this period but if this happens at
all it usually occurs later. The effects of water shock were therefore examined
after 3-5 h of culture. This schedule was also convenient because the pronuclei
have by this time become large enough to be clearly visible.
Eggs and their CFs may remain connected by a cytoplasmic stalk after treatment (Fig. 2) but they usually detach either while in water or during sucking
into the pipette.
Fig. 1 shows diagrammatically the types of eggs (denoted by Roman
numerals) produced by water shock, and their frequencies. Three types of
reaction in different combinations produced the types of eggs described:
68
JOLANTA OPAS
Table 1. Percentage reduction of egg volume after water shock {only
eggs from spontaneous ovulation)
Type of eggs and ploidy
Egg volume reduction*
II(2«)
IV(«)
V(2#i)
VI (3«)
Total
Mean±s.E.f
Maximal
No. of eggs and CFs measured
9-7 ± 6 1
25-4
72
19-5±2-8
380
13
16-1 ±1-9
34-1
21
9-7±0-8
24-8
60
ll-3±0 6
380
166
*
CF volume x 1QQ%
Treated egg volume+ CF volume
t Standard error.
Fig. 2. (A) Diploid egg of type V connected with its CF; left, the CF in focus; right,
pronuclei in focus. Arrow indicates the smaller pronucleus, which has probably
arisen from 2 P.B. nucleus. (B) Another example of CF connected with the egg.
Cytoplasmic stalk is clearly visible; note that a part of the pronuclear membrane is
still in the stalk. Differential interference contrast. Bar equals 20/tm.
(a) permanent incorporation of 2 P.B. and/or (b) escape of a CF and/or
(c) escape of a pronucleus (pronuclei) in the CF. Frequencies of these three
phenomena are also given in Fig. 1.
Character of CFs and reduction in egg volume
Table 1 gives the values of egg volume reduction for different types of eggs.
It is clear that both mean and maximal values are the highest in the eggs which
have produced a CF containing a pronucleus. CFs of this type are usually those
which start to form in the 1st to 3rd minute of treatment (see also p. 66), grow
rapidly and spontaneously stop enlarging. They usually consist of granular
Effects of low osmolarity on mouse zygotes
69
Table 2. Reaction of eggs to water shock in relation to the source of eggs
Source of eggs
I (In)
Induced ovulation
Spontaneous ovulation
Total
6-6
60
6-4
Type of eggs (%) and ploidy
*
No. of
11(2/7) III* IV (n) V (In) VI (3«) VII (3«) eggs
26-2
39-2
29-6
5-9
00
4-4
6-4
5-5
6-2
11-2
7-8
10-3
36-5
34-6
360
7-2
6-9
71
608
217
825
* Enucleated eggs.
Table 3. Reaction of eggs to water shock in relation to the post-fertilization stage
of eggs (only eggs from induced ovulation)
Type of eggs (%) and ploidy
Type of reaction (%)
A
2
CF
CF P.B.
ab- pres- with inof
1(2") II (In) I I P IV (/?) V (In) VI (3n) VII (3«) eggs sent sent PRt corp.
No. CF
Post-fertilization
stage of eggs
Pronuclei not seen
Pronuclei seen
Total
12-3
2-6
9-2
33-9
22-4
30-3
1-8
5-3
2-9
7-4
3-9
6-3
10-5
5-3
8-8
30-2
47-4
35-7
3-7
13 2
6-7
162
76
238
160
15-8
15-9
83-9
84-2
840
19-7 44-4
14-5 65.8
181 51-3
* Enucleated eggs, t PR, pronucleus.
cytoplasm and originate from eggs in which pronuclei could not be seen. On the
other hand CFs that start to form later and enlarge slowly, usually do not
exceed 5 % of initial egg volume. They contain 'hyaline' cytoplasm and originate
from eggs with large pronuclei.
Factors affecting the reaction of eggs
Source of eggs (spontaneous v. induced ovulation)
There is a statistically significant difference between the frequency of the
various egg types when eggs from spontaneous and induced ovulation are
compared (x2 = 24-25, P < 0-005) (Table 2). Diploids of type V, with a pronucleus replaced by 2 P.B. nucleus (Figs. 2 A, 3C), were more frequent among
eggs from induced ovulation. Enucleation (eggs of type III; Fig. 5) occurred
exclusively in this group. Diploids of type II were more common among
spontaneously ovulated eggs.
Age and post-fertilization stage of eggs
There were four groups of eggs, recovered between 18-5 h and 20 h after
HCG, at half-hour intervals. No significant differences in the frequency of egg
types resulting from water shock were found in the age range examined. Postfertilization stage of eggs does not necessarily correspond with their age,
70
JOLANTA OPAS
Fig. 3. Cytoplasmic fragment with a pronucleus (A), haploid egg with 2 P.B. present
(B) and diploid egg of type V (C). Compare the size of 2 P.B. nucleus (arrow) with
the size of the pronuclei of diploid egg, one of which has developed from 2 P.B.
nucleus. Differential interference contrast. Bar equals 20 /«n.
however. The eggs from any one female usually represent a wide range of
fertilization stages. For this reason the eggs were divided into those in which
pronuclei could be seen and those in which no pronuclei could be seen, and the
types of eggs produced within each group were compared (Table 3). There is
a statistically significant difference between the frequency of egg types in these
two groups (x2 = 21-65, P < 0-005). CFs containing pronuclei (Figs. 2, 3 A)
are produced more often from less advanced eggs, which results in a higher
frequency of haploids (Fig. 3 B) and diploids of type V in this group. Enucleation
is more frequent in the other group, however. Permanent incorporation of
2 P.B. is more common in more advanced eggs, leading to a higher incidence
of triploidy (Fig. 4). The percentage of eggs which have not produced a CF
remains at the same level irrespective of the post-fertilization stage of treated
eggs. There were many more diploids (type I) than triploids (type VII) among
eggs in which no pronuclei were seen, and many more triploids than diploids
among those with visible pronuclei.
Developmental capabilities of treated eggs
Some morulae and blastocysts developed from eggs with a known volume
reduction. These are listed in Table 4. The majority of eggs were not measured,
however. The development of these eggs is described below.
Diploid eggs of type I and II
Out of six unaffected eggs of type I transplanted, three blastocysts of about
100 cells each were obtained (Table 6).
Since the first transplantations of eggs of type II showed that they were also
able to develop into blastocysts and even beyond implantation, further
Effects of low osmolarity on mouse zygotes
71
Fig. 4. Triploid egg: left, one pronucleus in focus; right, other two pronuclei in focus
(the smaller of these two - indicated by an arrow - has probably developed from
2 P.B. nucleus). Differential interference contrast. Bar equals 20 /tm.
Table 4. Preimplantation development of eggs with known reduction of egg
volume
Type of egg
transplanted
and ploidy
IV (/i)
V(2«)
VI (3«)
Egg volume
reduction
Recovered embryos
Stage
Day
No. of cells
2 x blastocyst
Morula
Morula
2 x blastocyst
Blastocyst
5 and 11
4
11
19 and 24
7
26, 35
22
12
31, 29
43
Table 5. Preimplantation development of treated eggs
No. of embryos recovered
Type of
eggs and
ploidy
11 (2/i)
V(2/7)
IV («)
VI (3/i)
VI + VII (3«)
Total
No. of
eggs
transplanted
39
25
22
54
22
162
5th day
4th day
A
Morulae
1
Blastocysts
3
1
2
3
6
4
Morulae
Blastocysts
1
2
2
6
2
13
11
7
11
7
36
%of
Total
development
morulae + (morulae+
blastocysts blastocysts)
16
10
4
17
12
59
410
400
18-2
31-5
54-5
36-4
72
JOLANTA OPAS
Table 6. Number of cells of experimental embryos
No. of cells*
A
Type of
eggs and
ploidy
4th day
5th day
Blastocysts
Morulae
Morulae
Blastocysts
100, 102, 104
30, 32, 34
None
recovered
45
23
28,40
I {In)
II (2/i)
22
V(2/i)
None
recovered
12
IV (it)
VI (3n)
—
VII (3/i)
—
c
None
12, 22, 62
recovered
—
—
18, 25, 48
—
397 ±7-0
12-82(9)
52-2 ±10-6
26-76(6)
None
recovered
32-2 + 5.7
18-53(6)
59
None
recovered
* Mean number of cells ± standard error, variation and number of embryos (in parentheses)
are shown where there are more than three embryos examined in a given group. In the other
case number of cells of each embryo is shown.
Table 7. Postimplantation development of treated eggs
Type of eggs
transplanted
and ploidy
No. of
transplantations
No. of
eggs
No. of
implantations
No. of
embryos (%)
II (2/i)
V(2«)
VI + VII (3«)
6
5
6
21
25
18
11
0
6
11(52-3)
0
5* (27-7)
* Including three diploid embryos.
examination of the development of unaffected eggs (type I) was abandoned.
Out of 39 eggs of type II, 16 developed into morulae and blastocysts. Mean cell
number of blastocysts on the 5th day was 39-7 ±7-0, variation 12-82 cells
(Tables 5, 6).
Out of 21 transplanted eggs 11 embryos developed beyond implantation
(Table 7). Four recipients which had received nine eggs were killed between the
8th and 10th day of pregnancy and two embryos, retarded by one day but
otherwise normal, were recovered. One recipient was killed at term; out of four
transplanted eggs with known egg volume reduction (11, 15, 15, 16%) three
normal embryos developed, two of them being alive and one dead. One recipient was left till term and gave birth to six young from eight eggs transplanted,
four males and two females. All of them survived and proved to be fertile.
Effects of low osmolarity on mouse zygotes
73
Diploid eggs of type V
From 25 transplanted eggs, two morulae and eight blastocysts were obtained
(Table 5) but only three embryos yielded analysable metaphase plates. One
blastocyst found on the 4th day (Table 6) had a metaphase plate with T6 marker
and Ychromosome, which implies that the female pronucleus must have escaped
during water shock (but see also Discussion, p. 76). One morula and one blastocyst recovered on the 5th day were gynogenetic since they had no T6 chromosome
in the karyotype, hence the male pronucleus must have escaped in CF.
Blastocysts recovered on the 5th day had a mean cell number of 52-2 ± 10-6.
As shown in Table 7, no diploid eggs of type V developed beyond implantation. Transplantations were checked on the 8th, 9th and 12th day. In order to
increase the cell number at implantation eight eggs were first transplanted to the
oviducts of immature recipients, recovered in the morning of the 3rd day (one
4-cell and four 2-cell eggs) and retransplanted to two mice in the first day of
pseudopregnancy. Nevertheless, no implantation occurred.
Triploid eggs
Out of 76 eggs transplanted, 29 developed into morulae or blastocysts. Of
these transplants, 54 were of type VI only and the remainder were transplanted
as a mixture of types VI and VII. The only available blastocyst of type VII had
59 cells; type VI blastocysts had 32-2 ±5-7 cells on average (Tables 5, and 6).
From 18 tri-pronucleate eggs (Table 7), six decidual swellings containing five
embryos developed. Three embryos recovered on the 9th and 15th day of
pregnancy were diploid, and the remaining two were triploid. One triploid was
recovered on the 9th day and was a small egg-cylinder with no embryonic part
developed. Another, recovered on the 12th day, had beating heart, allantois,
yolk sac, but no head.
Haphid eggs {type IV)
Of 22 transplanted eggs four morulae were found (Tables 5, 6). In two cases
it was possible to examine the karyotypes: both embryos were gynogenetic
(no T6 marker). As no blastocysts were obtained, no attempt was made to
examine postimplantation development of haploid eggs.
Enucleated eggs (type III)
This was not a homogeneous group, despite what has been diagrammatically
shown in Fig. 1. Among 28 enucleated eggs 11 contained 2 P.B. (Fig. 5 A) and
17 did not. Among seven eggs of the latter type, in two cases 2 P.B. nucleus was
present in CF (CF thus contained three pronuclei). In five other cases 2 P.B.
nucleus was incorporated into the egg cytoplasm so that the eggs became
secondarily haploid. Neither the haploid eggs of this type nor the enucleated
ones ever divided regularly (two eggs fragmented).
74
J O L A N T A OPAS
B
Fig. 5. Enucleated egg with 2 P.B. (A) and its CF (B) in two focal planes to visualize
both pronuclei. Permanent preparation. Bar equals 20/tm.
Table 8. Preimplantation development of treated eggs in relation to the
source of eggs (eggs transplanted to immature females)*
Development (%)
A
Stage
recovered
Induced
ovulation
Spontaneous
ovulation
Total
Morulae and blastocysts
Cleaving eggs
1-cell eggs or empty zonae
All recovered!
No. of eggs transplanted
19-2
20-3
29-3
68-8
99
53-7
7-5
119
73-1
67
331
15-1
22-3
70-5
166
* Includes also eggs from mixed transplantations, for example of eggs of type IV + V not
shown in Table 4.
t 2-8-cell stage.
Comparison of the development of eggs from spontaneous and induced
ovulation
Marked differences have been observed between the percentage of preimplantation development of eggs from induced versus spontaneous ovulation.
As individual groups of eggs were small, the data for all types of eggs were
combined. The results are given in Table 8. Almost three times as many eggs
from spontaneous ovulation developed into morulae or blastocysts than from
induced ovulation. The percentage of all developing eggs (cleaving + morulae
+ blastocysts) was also higher among spontaneously ovulated eggs. The
differences are statistically significant (x2 = 22-38, P < 0-005). Similar differ-
Effects of low osmolarity on mouse zygotes
75
ences have been observed by Tarkowski (1977) in his study of the development
of egg halves. It looks as though the eggs from induced ovulation were more
susceptible to experimental treatment than spontaneously ovulated ones.
DISCUSSION
Prolonged culture (2-6 h) of unfertilized eggs in hypotonic media (3/5 and
4/5 dilutions) causes activation and suppression of 2 P.B. but promotes egg lysis
(Graham, 1971; Graham & Deussen, 1974). When Niemierko (personal communication) cultured newly fertilized mouse eggs in hypotonic medium
(3/5 dilution) in attempt to obtain genetic anomalies, the duration of treatment
sufficient to suppress 2 P.B. (about 1 h) caused degenerative changes in the egg
cytoplasm. Thus moderately hypotonic but prolonged treatment is more
harmful than drastic but short-lasting treatment.
Extremely low osmolarity induces two cytological effects, escape of CFs and
incorporation of 2 P.B., both of which could result from membrane extension
caused by rapid increase of egg volume. This membrane extension could supply
the CF with new surface area as well as broadening the connexion between the
egg and 2 P.B. It seems that the zona pellucida plays an important role in
producing both effects: it compresses 2 P.B. to the egg by resisting the pressure
of the swelling egg and it provides the sperm-slit as an outlet for the abundance
of swollen cytoplasm. The sperm-split, as described by Austin (1951), is
a narrow trace left in the zona pellucida by a penetrating spermatozoon. There
are three pieces of evidence that the slit through which the CFs escape is really
the sperm-slit: first, unfertilized eggs never produce CFs when placed in distilled
water, though they swell markedly (Opas, unpublished observations); second,
parthenogenetically activated eggs do not produce CFs though they swell and
can incorporate 2 P.B. (BaJakier, personal communication); and third, two
CFs very rarely escape from one egg, perhaps only when a supernumerary
spermatozoon has entered. These observations, and the fact that cytoplasmic
stalks are of similar width, suggest that CFs do not escape by bursting of the
zona pellucida due to increase of internal tension. It may be added that Austin
(1951) was able to displace rat egg cytoplasm through the slit by compressing
the egg with a cover slip.
The frequency of eggs which do not produce CFs remains at the same level
(about 15 %) irrespective of the source and post-fertilization stage of eggs.
Perhaps part of the egg surface (ca. 15 %) and/or cytoplasm has different
properties and is not able to expand through the slit.
The frequency of permanent 2 P.B. incorporation and the character of CFs
depend on the post-fertilization stage of eggs. In 44-4 % of eggs with pronuclei
too small to see under the dissecting microscope, and 65-8 % of eggs with larger
pronuclei, incorporation of 2 P.B. took place (Table 3). The re-extrusion of
2 P.B. may be more difficult when the connexion between the egg and 2 P.B.
76
JOLANTA OPAS
(mid-body, the surrounding membrane) has been destroyed in more advanced
eggs. In eggs with small pronuclei CFs usually start to escape earlier, enlarge to
a greater extent and are built of more granular cytoplasm than in the other
group. CFs originating from less advanced eggs contained one pronucleus in
17-9 % of cases and two pronuclei in 1-8 % of cases.The corresponding values
for CFs from more advanced eggs were 9-2 % and 5-3 % respectively (Table 3).
These differences suggest that some changes in the location of the pronuclei, or
in the mechanical properties of the egg, take place during the one-cell stage. In
sea-urchin eggs there is an increase of stiffness of the egg surface as well as of
the egg as a whole at fertilization, then a decrease followed by a subsequent
increase of both parameters before cleavage (Hiramoto, 1963, 1974).
Though the loss of a certain amount of cytoplasm influences the rate of
preimplantation development of treated eggs (diploid blastocysts of type II had
on the 5th day fewer cells than those developed from unaffected eggs), the loss
of up to 15 % of cytoplasm need not disturb normal development of diploid
eggs. The relation between the decrease of the egg volume and subsequent
development has not been systematically examined. Table 4 shows, however,
that the cell numbers attained by embryos do not seem to be correlated with the
amount of cytoplasm lost.
It has recently been shown (Tarkowski & Rossant, 1976; Tarkowski, 1977)
that haploid and diploid halves obtained by cutting fertilized mouse eggs
may develop into morphologically normal blastocysts. Comparison of development of haploid embryos obtained in this way with those produced by microsurgical removal of a pronucleus (Modliiiski, 1975) and by water shock shows
that only cutting yields blastocysts regularly. No blastocyst developed from
haploid eggs produced by water shock and Modliriski obtained only one
blastocyst among 36 multicellular haploid embryos. It may be that cutting is less
deleterious for eggs than sucking out pronuclei or inducing formation of CFs,
because the displacements of egg cytoplasm are small or none in the case of
cutting and more serious when a cytoplasmic flow is involved.
The results obtained show that water shock is a good method of producing
triploidy in the mouse. It yields more tri-pronucleate eggs (up to 60 %, Table 3)
and allows a higher percentage of preimplantation development (38 %, Table 5)
than the most effective method reported up to now (cytochalasin B; Niemierko,
1975). However, in some cases diploid embryos develop from tri-pronucleate
eggs, due to delayed re-extrusion of 2 P.B.
Diploid eggs with a male pronucleus replaced by 2 P.B. nucleus (type V) are
of great interest, as they might serve as control material for diploid parthenogenones arising by suppression of 2 P.B. The question of the ability of such
gynogenetic eggs to continue development beyond implantation remains open.
The small number of eggs transplanted and the fact that all of them originated
from induced ovulation might have been responsible for the negative results
obtained in this study.
Effects of low osmolarity on mouse zygotes
11
Water shock may also be used to produce enucleation. It is extremely
difficult to remove both pronuclei from fertilized mouse eggs microsurgically
(Modliriski, personal communication). By the production of cytoplasmic
fragments, sample of egg cytoplasm or a pronucleus may be obtained without
destroying the egg.
I am grateful to Professor Andrzej K. Tarkowski for many suggestions and encouragement
during the course of this work and for helpful criticism of the manuscript.
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AUSTIN,
{Received 10 June 1976)
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