J. Embryo!, exp. Morph. Vol. 30, 3, pp. 519-545, 1973
519
Printed in Great Britain
Parthenogenetic development of mouse embryos
in vivo
I. Prciniplaiitation development
By ANNA WITKOWSKA1
From the Department of Embryology, Zoological Institute,
University of Warsaw
SUMMARY
Newly ovulated eggs from CBA-p and CBA-T6T6 inbred strains were activated in situ
with an electric shock of 20-50 V. Both spontaneously ovulating females mated with
vasectomized males (strain A) and those induced to ovulate and kept apart from males were
used. The immediate reaction of eggs was examined 5-7 h after activation and their subsequent development was followed every day up to the 6th day (eggs examined on the 5th and
6th day were tube-locked by a ligature). In spontaneous ovulators the highest yield of activated eggs was obtained following 30 V shock (75 %), with a mean for four series (20, 30,
40 and 50 V) of 46-1 %. The most common reaction is extrusion of the second polar body
(2 PB) and formation of one pronucleus (about 70 % of activated eggs). Other types of
reaction include: immediate cleavage, suppression of 2 PB with formation of two or, very
rarely, one pronucleus. Thus the majority of eggs are potentially haploid. No relationship
between the frequency of various types of reaction and the strength of the shock (voltage)
was observed. Up to the 3rd day cleavage proceeds at a normal rate and appears to slow
down during the 4th day. In spontaneously ovulating females the incidence of blastocysts
among developing eggs rises from 3 4 % on the 4th day to 52-5 % on the 5th day; the
corresponding figures for induced ovulations are 10% and 23-6%. Some embryos despite
being composed of a large number of cells (40-120) do not undergo cavitation and remain
morulae.
On the basis of karyological examination of 60 embryos with analysable metaphase plates
60 % were classified as haploid, 16-7 % as diploid, 1-7 % as tetraploid and 21 -6 % as haploid/
diploid mosaics. During early cleavage binucleate blastomeres (a presumed step in regulation
from n to 2/?) were observed only in eggs from induced ovulations. In embryos developed
from spontaneously ovulated eggs njln mosaics were observed for the first time on the 5th
day. In general, spontaneously ovulated eggs developed better after activation (both in
quantitative and qualitative terms) than those obtained following hormonal treatment.
INTRODUCTION
It is a general experience of the students of early mammalian development
that unfertilized eggs can undergo spontaneous activation. Activation which in
the first instance expresses itself in the completion of the second meiotic division,
leads most often to fragmentation, normal cleavage being a rare event. Although
systematical studies of this phenomenon have not been carried out on many
1
Author's address: Department of Embryology, Zoological Institute, University of Warsaw,
00-927/1 Warsaw, Poland.
34
E M B
30
520
A. WITKOWSKA
species, there is ample evidence that eggs of various mammals differ as regards
the frequency of activation and their ability to undergo parthenogenetic development. In the hamster, for instance, as many as 80 % of eggs undergo activation,
and reform one or two pronuclei; some of these eggs can undergo first cleavage
(Austin, 1956). In the ferret as many as 30 % of eggs examined 48-66 h after
ovulation were at the 2- to 6-cell stage (Chang, 1950). Unfertilized eggs containing pronuclei were also observed in the rat (Austin & Braden, 1954a) but
the frequency of such eggs was low. Spontaneous activation occurs as a rule
in overripe eggs and is considered to be a result of changes taking place in the
cytoplasm and the nuclear apparatus prior to degeneration (Chang, 1950).
This is probably the main cause of the poor development of such eggs.
Eggs submitted to a parthenogenetic stimulus develop much more frequently
and attain more advanced developmental stages than the overripe eggs undergoing spontaneous activation. Artificial parthenogenesis has been studied in
several mammalian species, namely rabbit (Pincus, 1930, 1936, 1939 a, b;
Pincus &Enzmann, 1936; Pincus & Shapiro, 1940a, b; Shapiro, 1942; Thibault,
1947a, 1948, 1949; Chang, 1952, 1954), rat (Thibault, 1941b, 1949; Austin &
Braden, 1954a, b; Austin, 1956), mouse (Braden & Austin, 1954), ferret (Chang,
1950, 1957), sheep (Thibault, 1949; Thibault & Ortavant, 1949) and hamster
(Austin, 1956). In these experiments freshly ovulated eggs were subjected, in
vivo or in vitro, to the influence of various factors, e.g. cold shock, heat shock,
hypertonic and hypotonic solutions applied separately or successively, trypsin,
suspension of spermatozoa of other species, etc.
In many species low temperature proved to be a very effective parthenogenetic
agent. In the rabbit nearly 90 % of eggs exposed to the cold shock were activated
(Thibault, 1949), the percentage was about 60 in the ferret (Chang, 1957) and
nearly 80 in the hamster (Austin, 1956). Almost 100 % of rat eggs treated in
this way completed the second meiotic division (Thibault, 1947b, 1949), but
the reformation of a pronucleus was a very rare phenomenon (Austin & Braden,
1954a; Austin, 1956). On the other hand, mouse eggs are not activated under
the influence of cold shock (Braden & Austin, 1954). In these early studies
parthenogenetic morulae and blastocysts were obtained only in the rabbit
(Pincus & Shapiro, 1940a, b; Shapiro, 1942; Thibault, 1947a, 1949; Chang,
1954) and in the ferret (Chang, 1957).
The most frequent reaction of eggs to parthenogenetic agents is the extrusion
of the second polar body (2 PB); this can be accompanied by reformation of
a haploid pronucleus. Completion of the second meiotic division can also take
place without extrusion of 2 PB and it then results in the formation of a single
diploid pronucleus, or two haploid pronuclei, or immediate cleavage. Sometimes chromosomes become scattered in the cytoplasm as a result of the shock
and more nuclei of different sizes are formed. Similarly as in the case of spontaneous activation many of the artificially activated eggs are soon subjected to
fragmentation or stop developing during cleavage.
Preimplantation parthenogenetic development
521
The ploidy of artificially activated eggs may be determined immediately after
activation on the basis of the number of pronuclei and the presence or absence
of second polar body. Information regarding the ploidy of developing embryos,
especially blastocysts, is, however, of greater value. In the rabbit chromosomal
studies were only carried out by Chang (1954) who found that parthenogenetic
blastocysts were diploid. According to Chang (1954) cooling of eggs causes
disappearance of the fibres of the spindle which results in the formation of one
diploid pronucleus. Braden & Austin (1954) counted chromosomes in two
2-cell mouse eggs on 'squash' preparations and found that the eggs were haploid.
Earlier studies have not provided much information on the parthenogenetic
development of mouse eggs. Braden & Austin's (1954) report was the only one
published on this subject. They found that eggs submitted to the temperature
of 44-45 °C reacted either by extrusion of 2 PB and formation of one pronucleus or by undergoing immediate cleavage (division of a secondary oocyte into
two cells of equal size). Development of these eggs was not traced beyond the
4-cell stage. Recently parthenogenetic development of mouse eggs to the blastocyst stage was obtained after activation with electric current (Tarkowski,
Witkowska & Nowicka, 1970) and as a result of culturing cumulus-free eggs
in vitro (Graham, 1970).
The present study describes in detail the preimplantation development of
mouse eggs in vivo following electric stimulation. Preliminary results of some of
the experiments described here were presented previously (Tarkowski et al.
1970).
MATERIALS AND METHODS
Females of the CBA-p and CBA-T6T6 inbred strains were used throughout
the experiment. Eggs were obtained from spontaneous or induced ovulation.
In the first case females were left overnight with vasectomized males of A strain
and the activation was carried out in the morning, between 8 and 10 a.m. In the
case of CBA-T6T6 females (two marker chromosomes in the karyotype) it was
possible to verify the sterility of males by means of the chromosomal examination of embryos. Females in which ovulation was induced (5 i.u. of PMS followed after 34-35 h by 5 i.u. of HCG) were kept apart from males. Activation
was carried out 14-16 h after HCG injection.
The activation procedure was described previously (Tarkowski et al. 1970);
in short, it consisted in applying to the ampulla an electric shock produced
from an 8 /i¥ capacitor charged through a rectifier from the a.c. source of a
known voltage. In the series of experiments in which eggs were recovered on
the first day (5-7 h after activation) shocks of 20, 30, 40 and 50 V were applied;
in the series checked on the second and third days only a 40 V shock was used
and in the series checked on the fourth, fifth and sixth days stimulation was
carried out mainly at 40 V but occasionally also at 30 V. The operations were
performed under Nembutal anaesthesia. In earlier experiments eggs were
34-2
522
A. WITKOWSKA
Preimplantation parthenogenetic development
523
activated only in one oviduct, the other side serving as control. As no activation was observed on the non-operated side (see Results), some of females were
operated on both sides.
On the successive days of development eggs were flushed from the oviduct
or uterus and either permanent preparations were made from them (a modified
technique of Mulnard: Tarkowski & Wroblewska, 1967; Tarkowski, 1971), or,
in the case of morulae and blastocysts, air-dried preparations (technique of
Tarkowski, 1966). Prior to making chromosome preparations, colcemid was
injected to some of females (0-002 mg/g body weight) about 2 h before killing.
RESULTS
First day
As a result of the shock four kinds of eggs were obtained: non-activated,
activated, fragmenting and degenerated. The non-activated eggs contained
intact metaphase plate (Fig. 1), or, rarely, chromosomes were scattered in the
cytoplasm. Not a single egg obtained from oviducts on the non-operated side
showed any signs of activation.
In activated eggs the completion of the second meiotic division can take
place with or without extrusion of the second polar body (2 PB). In the first
case one pronucleus is reformed (Fig. 2). When 2 PB is suppressed the egg can
contain two pronuclei (Fig. 3), one pronucleus (Fig. 4), many small nuclei
(Figs. 6, 7), or it can undergo immediate cleavage (Fig. 5). The formation of
pronuclei was already observed 3 h after the shock. The majority of eggs were
examined 5-7 h after the shock, i.e. when pronuclei were already very big.
The identification of polar bodies did not present any difficulties. In the first
polar body chromosomes are usually scattered and the body soon degenerates.
On the contrary, 2 PB as a rule contains a nucleus and is usually retained up to
late cleavage stages.
FIGURES 1-8
Fig. 1. Non-activated egg with the spindle of the second meiotic division, 1st polar
body. Spontaneous ovulation. x 700.
Figs. 2-7. Activated eggs. First day of development, x 700.
Fig. 2. Egg with one pronucleus and 2nd polar body. Induced ovulation.
Fig. 3. Egg with two pronuclei; no 2nd polar body. Induced ovulation.
Fig. 4. Egg with one pronucleus, supposedly diploid; no 2nd polar body. Spontaneous ovulation.
Fig. 5. Egg which underwent immediate cleavage. Spontaneous ovulation.
Fig. 6. Egg with several nuclei; no 2nd polar body. Induced ovulation.
Fig. 7. Egg with one big pronucleus and a small subnucleus; no 2nd polar body.
Induced ovulation.
Fig. 8. Normal 2-cell egg with 2nd polar body; second day of development, x 700.
12$
9%
30
40
50
37
6}
20
Total
36
iot
Voltage
1000%
130
1000%
29
1000%
41
1000%
24
100-0 %
Total
no. of
eggs
No. of
oviducts
22-3%
29
3-4%
1
31-6%
41
58-6%
17
43-9%
18
16-6%
8 • *\ o /
0
4
5-5 V
2
2
72-2%
26
461%
1000 %
60
380%
1000 %
11
56-1%
1000%
23
750%
1000%
18
22-2%
1000%
8
no.
Total
1-7
1-2
2-1
30
0-8
Mean no.
per oviduct
750%
45
63-6 %
7
740 %
17
77-7 %
14
87-5 %
7
00
2 P B absent
1
o/
100%
6
91 %
0.7
2
16-6%
3
0
100 %
91%
6
1
13-0%
3
5-7 °/
1
12-5%
1
1-7%
1
91 %
1
0
0
0
Immediate
cleavage 2 pronuclei > 2 nuclei
(2/7)
(2/0
00
* 2 PB = :Second polar body.
t All females operated bilaterally.
$ All eggs recovered from control oviducts were non-activated (18 oviducts, 64 eggs).
3-55
3-2
3-4
40
3-6
Mean no.
Nonof eggs per activated ]Degenerated
oviduct
eggs
eggs
2PB*
present
1 pronucleus
Activated eggs
Table 1. Reaction of eggs 4-10 h after activation; spontaneous ovulation - vasectomized male
o/
/o
2 /o
J
J-1.1
J
y
9-1 °/
1
4-3 %
1
0
0
1 pronucleus
(2/0
O
KJ
Total
Inducedf
Spontaneous*
Type of
ovulation
Total
30
40
fO
Total
:o
20
30
40
Voltage
119
6
43
10
59
8
18
23
11
CO
Total no. of
activated eggs
/o
83
4
25J
9
38
7
14
17
7
45
7
0
1
0
1
0
3
2
1
6
Immediate
cleavage
29
33-9 %
2
17
1
20
15 0%
1
1
4
3
9
Total no.
c
75-6 %
24-4 %
* Data from Table 1.
! Data from Tarkowski et al. (1970).
% Three eggs contained in addition small subnuclei.
§ Two eggs (contained in addition small subnuclei.
90
66-1%
4
26
9
39
°- > u
f> N ft ° /
7
17
19
8
51
Total no.
1 pronucleus
+ 2PB
A
Haploids
Table 2. Ploidy of activated 1-cell eggs
17
1
10
0
11
1
1
3
1
6
2 pronuclei
c
7
1
4
1
6
0
0
0
1
1
> 2 nuclei
A
2 P B absent
Diploids
5
0
3§
0
3
0
0
1
1
2
1 pronucleus
• \
-5
3
#^
<-i
3
3
O
3
£5
g
3
1"*
"5
526
A. WITKOWSKA
Spontaneous ovulation {Tables 1 and 2)
There is a clear correlation between the voltage of the shock and the incidence
of non-activated, activated and degenerated eggs. With stronger current the
percentage of non-activated eggs decreases while the number of degenerated eggs
increases. The highest yield of activated eggs was obtained following 30 Y shock.
Among the activated eggs those with a single pronucleus and 2 PB (potentially haploid) predominated. As eggs which undergo immediate cleavage are
also haploid, the haploid eggs constituted altogether 85 % (Table 2). Potentially
diploid eggs, i.e. eggs in which the whole nuclear apparatus was retained (two
haploid pronuclei, one diploid pronucleus, many nuclei) accounted for only
15 %. No relationship between the frequency of various types of reaction and
the strength of the shock (voltage) was observed.
Induced ovulation (Table 2)
The results of this series of experiments were presented previously (Tarkowski
et ah 1970) but they deserve a short comment. In general the proportion of activated eggs was slightly lower at all voltages than among eggs from spontaneous
ovulation, the most effective stimulation being at 40 V rather than at 30 V.
This was most probably due to larger number of eggs and, consequently, larger
size of ampullae in superovulated females.
Haploid eggs (2 PB, one pronucleus) accounted for only 64-4 % of activated
eggs. Eggs which underwent immediate cleavage were very rare. On the contrary,
diploid eggs (2 PB suppressed) were encountered more frequently; the majority
of them contained two pronuclei. Eggs which contained more than two nuclei
and eggs in which one or two pronuclei were accompanied by small subnuclei
were observed also more frequently than in spontaneously ovulating females
(Fig. 7). It is not known whether subnuclei contribute chromosomes to the
spindle of the first cleavage and, therefore, whether in such eggs euploidy can
be restored.
Second day
Spontaneous ovulation {Table 3)
Among ten developing eggs there were one 1-cell egg with two nuclei and
2 PB, seven 2-cell eggs and two 4-cell eggs. The 1-cell egg reacted probably to
the shock by extrusion of 2 PB and formation of a haploid pronucleus but the
first cleavage was not completed, i.e. karyokinesis was not followed by cytokinesis. Two-cell eggs looked quite normal (cf. Figs. 8, 9). Out of the seven
2-cell eggs, 2 PB was found in six, which proved that they were haploid. One
4-cell embryo lacking 2 PB must have developed from an egg which had undergone immediate cleavage. The origin and ploidy of another 4-cell embryo in
which 2 PB was present, is unknown.
5
23
39
2nd
3rd
Total
122
66
17
39
Total
no. of
eggs
30
2-9
3-4
3-5
o
7
5-8%
y z. /
1
5-8%
6
9-2 °/
0
1-cell
J
/
o
7
5-8%
/
o
39
31-9%
z.o o /
J
/o
69
56-5%
Jt
—
—
10 (6)
3 (1)
—
2(1)
0
4-cell
7(6)
2 (0)
2-cell
A
Developing eggs
16
23
21 (17)t
410% 590%
2
4
10
1 (1)
11-8% 23-5% 58-8%
5
19
36
0
7-5 °/ 28-8 °/ 54-5 V
0
Mean
no. of
NonFrag- Degeneeggs per activated menting rated
,
oviduct
eggs
eggs
eggs Total no.
—
23 (3)J
0
0
N
± 8-celI
88
30
18
40
Total
no. of
eggs
3-4
3-3
3-5
3-3
i\> u /o
*-J
^ /o
40
0
1000%
18
0
1000%
23
7
76-6 V 23-4 °/
Mean
no. of
NonFrageggs per activated menting
oviduct
eggs
eggs
Non-operated side (control)
^
|*
<^>
«?.
§
Sg
c|
|
^
Sj.
^
S"
J^
bo
5S
t In parentheses number of eggs with 2 PB.
^
% Thirteen eggs were studied in the living state only and no information is available regarding the presence (or absence) of the 2 PB; 3 out of 10 eggs mounted s
in permanent preparations contain 2 PB.
f^
* Data from Table 1.
11
1st*
Day of
develop- No. of
ment oviducts
Operated side
Table 3. Development of activated eggs during the first 3 days; spontaneous ovulation - vasectomized male {voltage: 40 V)
528
A. WITKOWSKA
Induced ovulation (Table 4)
Out of 145 eggs, 52 (35-9 %) were activated. The majority of them (48) were
2-cell, and 36 had 2 PB. Cytoplasmic fragments were present in the perivitelline
space of almost all (45) 2-cell eggs (Fig. 10). 2-cell eggs without 2 PB which accounted for one third of all 2-cell eggs, developed probably from eggs equipped
with two pronuclei (Table 2). If this was the case, they were diploid.
Out of three 1-cell eggs, one had 2 PB and the spindle of first cleavage, one
contained two pronuclei and a small subnucleus and another one had four
nuclei and many cytoplasmic fragments.
A single 4-cell egg lacked 2 PB and was composed of two larger and two
smaller blastomeres and several cytoplasmic fragments.
In comparison with the first day the incidence of non-activated eggs decreased
while that of fragmenting eggs increased. It is worth noting, that on the second
day a similar percentage of fragmenting eggs was observed also in control
oviducts.
Third day
Spontaneous ovulation {Table 3)
The majority of embryos were in the 4-cell and 8-cell stage. There were also
three 2-cell, four 5- to 7-cell eggs and two 12-cell eggs. Morphologically the eggs
looked normal.
In six out of ten 4-cell embryos 2 PB was present, so they could not develop
from eggs which had undergone immediate cleavage. Among ten approximately
8-cell eggs out of which permanent preparations were made, 2 PB was found only
in three eggs. The low incidence of eggs with 2 PB as compared with the first
and second day suggest that by the third day 2 PB disintegrates in some of the
FIGURES 9-15
Fig. 9. Regular 2-cell parthenogenetic egg; second day of development. 2nd polar
body present but out of focus. Spontaneous ovulation. x 700.
Fig. 10. Three parthenogenetic 2-cell eggs; second day of development. Two eggs
contain cytoplasmic fragments in the perivitelline space. Induced ovulation.
x400.
Fig. 11. Four-cell parthenogenetic egg; third day of development. Blastomeres are
regular, 2nd polar body present. Induced ovulation. x 700.
Fig. 12. Normal 4-cell egg with 2nd polar body. Shown for comparison with
parthenogenetic eggs, x 700.
Fig. 13. Two-cell parthenogenetic egg; third day of development. Each blastomere
contains two nuclei. Second polar body absent. Induced ovulation. x 700.
Fig. 14. Three-cell parthenogenetic egg; third day of development. The larger blastomere contains two nuclei. Second polar body present. Induced ovulation. x 700.
Fig. 15. A small parthenogenetic blastocyst composed of 24 cells; 2nd polar body
absent. Fourth day of development. Spontaneous ovulation. x 600.
Preimplantation parthenogenetic development
529
14
15
17
16
42
2nd
3rd
Total
346
106
145
95
8-2
6-6
8-5
10 5
Fragment Degene,
ing
rated
Total
eggs
eggs
no.
1-cell
2-cell
26
7-5%
68
115
137
19-6% 33-2% 39-6%
—
—
—
7 (4)
0
0
3-cell
Developing eggs
17
6
29
43 42(24)t 1 (0)
17-9%
6-4% 30-5% 45-2%
7
27
59
52
3(1) 48(32)
4-8% 18-6% 40-7% 35-9%
2
35
27
42
0
9 (3)
1 -9 V 33-0 V 25-4 V 39-6 °/
Nonactivated
eggs
* Data from Tarkowski et al. (1970).
f In parentheses number of eggs with 2 PB.
9
1st*
Day of No. of
develop- oviment
ducts
Mean
no. of
Total
eggs
no. of
per
eggs oviduct
Operated side
—
25 (14)
1 (0)
0
4-cell
—
1 (1)
0
0
24
4
11
9
—^
No. of
> 4ovicell
ducts
180
36
68
76
7.5
90
6-2
8-4
Mean
no. of
Total
eggs
no. of
per
eggs oviduct
Fragment- Degeneing
rated
eggs
eggs
H
76
0
0
1000 %
O
56
12
0
82-3% 17-7%
14
15
7
38-9% 4 1 - 6 % 19-5%
.
Nonactivated
eggs
Non-operated side (control)
Table 4. Development of activated eggs during the first 3 days; induced ovulation, no male {voltage: 40 V)
O
Preimplantation
parthenogenetic
development
531
parthenogenetic embryos and, consequently, that these data are not indicative
of the high incidence of diploids.
The relatively large number of eggs with more than four blastomeres (23 out
of 36) provides evidence that during the first 48 h of development parthenogenetic eggs cleave at a rate characteristic for fertilized eggs (in CBA strains the
embryos on the third day are in the 4- to 8-cell stage, the 8-cell eggs predominating only in the afternoon). On this day for the first time fragmenting eggs were
found in control oviducts.
Induced oxidation {Table 4)
Out of 42 developing eggs 25 attained the 4-cell stage. Some 2-, 3- and 4-cell
eggs had binucleated blastomeres, for instance: 2-cell eggs with either one or
both binucleated blastomeres (Fig. 13), 3-cell eggs in which larger blastomere
had two nuclei (Fig. 14) and 4-cell eggs containing one binucleated blastomere.
Binucleated blastomeres must have arisen as a result of nuclear division which
was not followed by cytokinesis. Embryos with binucleated blastomeres
accounted for about 30 % of developing embryos. It should be emphasized that
this phenomenon has never been observed at this developmental stage in
embryos recovered from spontaneously ovulating females.
The increase in the number of fragmenting eggs on the operated side must
have been mainly due to fragmentation of non-activated eggs (cf. control oviducts-from the 2nd to the 3rd day fragmenting eggs increase from 17% to
41 %)•
Fourth day
Spontaneous oxidation {Table 5)
Among 58 developing embryos which constituted 65-6 % of recovered eggs,
morulae (embryos composed of at least 8 cells) were most numerous (89-8 % of
developing eggs). There were also four cleaving embryos clearly retarded in
development (one 2-cell and three 4-cell) and two blastocysts. The average number of morulae and blastocysts per uterine horn was 1-71, which is more than
a half of recovered eggs.
The cell number of 13 morulae varied from 8 to 30, with a mean number of
18-2 (Fig. 24, Table 7). Ploidy was determined in seven morulae: six were
haploid and one diploid. The only two blastocysts recovered on the fourth day
proved to be diploid.
Induced ovulation (Table 6)
Only ten developing embryos were found among 57 eggs recovered from four
uterine horns and six oviducts. These comprised: one cleaving egg, eight morulae
and one early blastocyst. Chromosomal preparations were made from two
morulae: one was built of eight cells and had three haploid metaphase plates,
another one had 21 interphase nuclei and one tetraploid plate (Figs. 18, 19). In
the latter embryo 2PB could not be detected. The blastocyst was built of
151
460 %
120
50-4 °/
67
56-8 %
53
440'
31
34-4
Nondeveloping
eggs*
176
54 0 %
1000 %
43-2 %
1000%
118
49-6%
100 0 %
67
560%
100 0%
51
58
65-6 %
1000%
Total
no.
lOOJ
57-0%
6-9%
40-7%
6-8%
12
43-1%
48
22
JO 8 / 0
7-9%
8
4
O U /o
89-8^
6-8 °A
26
52
Morulae
5= 8-cell
4
Cleaving
eggs
2-7-celI
36-1%
64
52-5%
490%
62
25
33-Z / o
37
3-4
1-49
1-42
1-26
1-54
1-71
0-78
0-66
0-90
006
Mean no. of
morulae and Mean no. of
blastocysts blastocysts
Blastocysts per oviduct per oviduct
* This group comprises non-activated, fragmenting and degenerated eggs.
t All eggs from ligated oviducts.
% Thirty-three eggs inspected in the living state only (used for transplantation); air-dried preparations made from 13 eggs and total preparations from 54 eggs.
2-94
327
1000%
Total
111
79
30
Total
316
118
1000%
38
40
5thf
3 00
2-85
120
1000%
41
40
4th
238
1000%
2-78
89
1000 %
32
Voltage
Day of
development
Mean
no, of eggs
per oviduct
Total
no. of
eggs
Total
no. of
oviducts
Developing eggs
Table 5. Parthenogenetic embryos on the 4th and 5th day of development; spontaneous ovulation- vasectomized male
(75
O
H
K)
Total no.
of oviducts
10
30
4
44
Day of
development
4th
5tht
6thf
Total
1000%
333
1000%
286
85-8%
47
12-5%
3
13-5%
100 0%
34
17-6%
1000 %
10
no.
Total
17
0
44-1%
15
200%
2
Cleaving
eggs
2-7-cell
18
0
32-3%
11
700%
7
Morulae
^8-cell
14-2 %
1000%
36-1 %
38-3 %
* This group comprises non-activated, fragmenting and degenerated eggs
t All eggs from ligated oviducts.
7-57
87-5 %
21
600
24
218
86-5 %
8-40
47
82-4 %
5-70
1000%
252
1000%
57
Total no.
of eggs
Nondeveloping
eggs*
Mean no.
of eggs
per oviduct
25-6 %
12
3
23-6 /0
8
100%
1
Blastocysts
Developing eggs
0-68
0-75
—
0-75
0-26
010
0-80
0-63
Mean no. of
blastocysts
per oviduct
Mean no. of
morulae and
blastocysts
per oviduct
Table 6. Panhenogenetic embryos on the 4th, 5th and 6th day of development; induced ovulation, no male {voltage: 40 V)
534
A. WITKOWSKA
18
20
22
23
Preimplantation parthenogenetic development
535
24 cells and also lacked 2 PB (Fig. 15). The developing embryos accounted for
17-6 % of eggs only, while on the third day as many as 40 % were classified as
developing (Table 4). Such a sudden decrease in the number of developing eggs
between the 3rd and 4th day has not been observed in females which ovulated
spontaneously and were pseudopregnant.
Fifth day
Immediately after applying the electric shock, oviducts were ligated at the
tubo-uterine junction to prevent passage of eggs to the uterus and thus to prolong
their preimplantation development. On the 5th day oviducts were rinsed and
air-dried preparations were made from morulae and blastocysts.
Spontaneous ovulation (Table 5; Fig. 24)
Morulae and blastocysts accounted for over 90 % of developing eggs, the
remaining ones being retarded in cleavage. Air-dried preparations were made of
19 morulae and 30 blastocysts. Both morulae and blastocysts had on the average
a high number of cells (nuclei), with a mean of 54-8 and 72-0, respectively. In
normal CBA embryos cavitation begins at a stage of about 32 cells. In many
of the parthenogenetic embryos blastulation was clearly delayed - out of
19 morulae 15 had more than 32 cells. It is worth noting that a blastocoelic
cavity was not present even in some embryos composed of as many as
FIGURES
16-23
Fig. 16. A small mosaic blastocyst with one not incorporated blastomere; fifth day
of development. Spontaneous ovulation. x 600.
Fig. 17. Irregular haploid blastocyst; fifth day of development. Spontaneous ovulation. x 600.
Fig. 18. Air-dried preparation of a 3-5-day-old regular parthenogenetic morula of
CBA-T6T6 origin: 21 nuclei, varying in size and one tetraploid metaphase plate.
Induced ovulation. x 270.
Fig. 19. Part of Fig. 18 under higher magnification. Tetraploid metaphase plate
(80 chromosomes); four marker chromosomes are marked with arrows. x700.
Fig. 20. Air-dried preparation of a mosaic blastocyst; fifth day of development.
In a living state the blastocyst was described as large and regular. There are two
diploid and three haploid metaphase plates. Note great differences in the size of
nuclei. The nucleus of the 2nd polar body is marked with an arrow. Spontaneous
ovulation. x 190.
Fig. 21. Part of Fig. 20. Diploid metaphase plate with two marker chromosomes
(arrows), x 640.
Fig. 22. Haploid metaphase plate from a haploid blastocyst; one marker chromosome (arrow). Fifth day of development. Spontaneous ovulation. x 670.
Fig. 23. Haploid and diploid metaphase plates from a mosaic blastocyst. Marker
chromosomes are marked with arrows. Fifth day of development. Spontaneous
ovulation. x700.
35
EMB
30
40
10
2U2
> b
> )
>> >
s
10
20
30
40
50
60
70
80
90
100
no
-:
-;
" ':
•;
-i
-i
-;
-I
:
;
:
;
if
! :
! i
; ;
fl
: 2
'. yS
:
\
?S
*.
.
.'.
:
:
: :
.
M M ! 1 II
! ; MM
• • ?: $ ? > I
j : S J!j •
: HI In
-: : : !
PI •
• %%> ) S •>
• \ 1) ) \ ' \
MM
-
10
• 1 1 s 1 1 '• 2Io n 3 0
50
60
70 -
80 -
10
20
30
40 -
p
5
j
?
>
}
)
lira
Induced
ovulation
Fig. 24. Number of nuclei (including number of metaphase plates) and ploidy of parthenogenetic embryos on the 4th and 5th day of development
10
< 20 -
>> spn
| No. of metaphase plates
[] Unknown ploidy
g] Mosaic
ra
Q Haploid
0 Diploid
[[] Tetraploid
inn.
10
20
30
50
50 -
60 -
- :: 1
1m
70 -
: : :
70 -
60
80 -
Spontaneous
ovulation
5th day
80
30
20
5 30 _
CO
I
!
-
90 -
100
no
40
Induced
ovulation
120 -
40
Spontaneous
ovulation
4th day
O
H
>
O\
Preimplantation parthenogenetic development
537
Table 7. Number of cells and metaphase plates in control and parthenogenetic
morulae and blastocysts {activation at 30 and 40 V)
Control
embryos*
Day of development...
4th
Parthenogenetic embryos
f
4th
5thi
A
Type of ovulation
No. of embryos
Morulae
Blastocysts
Total
Spontaneous
Induced
13
2
15
4
1
5
309
185
494
Mean no. of cells
Morulae
Blastocysts
No. and % of embryos
with metaphase plates
Morulae
Blastocysts
—
18-2
32-5
23-8
30-7
233
75-4%
133
71-9%
9
69-2 %
2
1000%
Mean no. of metaphase plates
per embryo containing dividing cells
Morulae
3-7
2-3
10
Blastocysts
4-3
2-2
Total
40
Spontaneous
Induced
Total
19
39
58
7
5
12
43
47
—
11-5
240
54-8
720
25-4
50-4
—
—
2
50-5 %
0
—
16
84-2 %
34
87-2 %
6
85-7 %
4
800%
33
—
40
—
2-5
—
2-5
3-5
5-6
4-9
4-5
7-7
5-8
—
—
—
* Data from Tarkowski & Jendyk (in preparation).
50-100 cells. Similarly as in normal embryos mitotic activity (number of metaphase plates per embryo) was higher in blastocysts than in morulae (Table 7).
Chromosomal studies have shown that some embryos contained only haploid
metaphase plates (Fig. 21), other only diploid plates (Fig. 22), and still other
were nJ2n mosaics (Fig. 23). Two blastocysts shown in a living state in Figs. 16
and 17, proved to be mosaic and haploid respectively.
Induced ovulation {Table 6; Fig. 24)
As many as 252 eggs were rinsed from the ligated oviducts but only 34
(13-5 %) could be classified as developing: nearly half of these embryos were
retarded in cleavage. The mean number of morulae and blastocysts per oviduct
was much lower than in spontaneously ovulating females (0-63 versus 1-42) in
spite of the fact that the total number of eggs was on the average much higher
(8-4 versus 3-0). The average number of cells was lower than in embryos developing from spontaneously ovulating eggs (Table 7).
35-2
538
A. WITKOWSKA
Table 8. Ploidy of parthenogenetic embryos*
(activation at 30 and 40 V)
No. of embryos> and ploidy
Day of
development
n
Type of
ovulation
Spontaneous
Induced
5th
Spontaneous
Induced
Total
4th
In
I7/2/7
,—*-—\
t— - *
»
Total
An
,—*-—^
,—^-
r—•
Mf
BJ
M
B
M
B
M
B
M
B
6
1
7
2
16
0
0
20
0
20
0
0
4
0
4
0
0
8
1
9
1
0
3
0
4
2
0
3
1
6
0
1
0
0
1
0
0
0
0
0
7
2
14
2
25
2
0
31
2
35
'
*•
*•
r—'
v
v-
-v
V ~
10
13
211-6%
600 V
16-7 V
/o
/o
* A part of these data was published in Tarkowski et al. (1970).
t M = morulae.
% B =blastocysts.
36
v
—V-
1
1-7!Vo
60
1000 %
Sixth day
Induced ovulation
Among 24 eggs recovered from four ligated oviducts only three were developi n g - all were blastocysts composed of ca. 150, 65 and 30 nuclei. The blastocyst
with a lowest number of cells was described in a living state as a trophoblastic
vesicle.
DISCUSSION
Electric shock applied to the ampulla is a very effective parthenogenetic agent:
at optimal voltage over 50 % of eggs are activated and the majority of them
develop into multi-cellular morulae or blastocysts (Tables 1, 5). A very high
percentage of activation (about 65 %) can also be obtained as a result of in vitro
culture of cumulus-free eggs (Graham, 1971). In earlier studies by the same
author (Graham, 1970) about 40 % thus activated eggs attained the stage of
morula or blastocyst. By submitting mouse eggs to the temperature of 44-45 °C
in vivo (Braden & Austin, 1954) or in vitro (Komar, 1973) it is also possible
to activate about 50 % of eggs, but it seems that such embryos have poor
developmental prospects. Braden & Austin (1954) did not find embryos with
more than four blastomeres and Komar observed that only 5 % of eggs developed as far as to the blastocyst.
In all the above experiments the reformation of pronucleus or pronuclei was
taken as a criterion of activation. The extrusion of the second polar body is not
by itself a sufficient criterion, as it is not always followed by reformation of
a nucleus in the egg. For instance, rat eggs submitted to cold shock extrude
2 PB in almost 100 % of cases, but only very few of them reform the pronucleus
4th
5th
1000%
Morulae
Mean
no. of
No. nuclei No.
—
—
—
-
—
761
—
—
27-7!
0
13
—
—
-
-
-
Morulae
Diploids
Blastocysts
—
10
—
-
29-3
45-8
12-7% -
3
-
-
1
230
3
36-3
21-5 °/
2
3
32-5
55-5
Mean
Mean
Mean
Mean
no. of
no. of
no. of Total no. of
No. nuclei no. nuclei No. nuclei No. nuclei
Blastocysts
67-9
63-6
3 49-3
— 21-5% —
—
—
—
—
— 30-3% -
—
Mean
Mean
Mean
no. of
no. of Total no. of
nuclei no. nuclei No. nuclei
Blastocysts
Morulae
230
6
230
0
750
8
700 20
— 570% —
—
—
—
— 60-5%
59-6% —
7
2
6
14
33
28
1000%
—
— 1000% —
Total no. of
Day of
embryos
Mean
de,
* »
velop- Moru- Blasto- Total no. of
ment
lae cysts no. nuclei
Mosaics
Haploids
Table 9. Number of cells {nuclei) in 4- and 5-day parthenogenetic embryos depending on developmental stage and ploidy;
spontaneous ovulation - vasectomized male
vo
a
a
a
f
540
A. WITKOWSKA
and undergo first cleavage (Thibault, 1949; Austin & Braden, 19546; Austin,
1956). Hamster eggs react in a similar way: under the influence of a cold shock
about 80 % of eggs are activated, but despite the extrusion of 2 PB the reformation of the pronucleus does not occur in all eggs. However, the formation of
a nucleus as a result of activation, although it is a prerequisite of further development, does not yet indicate that the egg is able to complete the whole
preimplantation development. The viability of artificially activated eggs must
depend also on other factors (probably cytoplasmic in nature) and differ
from egg to egg - some stop developing during early cleavage, others develop
as far as to the blastocyst.
Differences in the effect of various stimuli which can be observed at the onecell stage, are rather of quantitative nature and are concerned with:
(1) Regularity of the second meiotic division, i.e. formation of euploid pronucleus (pronuclei) versus aneuploid pronucleus (pronuclei) accompanied by
subnuclei. The formation of subnuclei is a rare phenomenon after electric stimulation and occurs frequently after heat shock (Komar, 1973). The presence
of subnuclei may result in aneuploidy which can be an additional obstacle in
the development of parthenogenetic embryos, especially hypohaploid.
(2) Frequency of various types of reaction (extrusion of 2 PB, suppression
of 2 PB, immediate cleavage). For instance, it seems that culture in vitro and
heat shock cause immediate cleavage more often than electric stimulation. However, the validity of such comparison is open to criticism because animals used
in various experiments were genetically different. It is possible therefore that
the differences observed have also a genetic background and should not be
attributed solely to the type of parthenogenetic agent. This finds support in the
fact that eggs of A strain stimulated electrically react more often by formation
of two haploid pronuclei (Tarkowski et ah 1970) than CBA eggs (present study).
It is known that various abnormalities in the meiotic divisions of the oocyte
occur with different frequency in different strains (Braden, 1957). The frequency
of these abnormalities may be increased when eggs are activated by an artificial
stimulus rather than a spermatozoon. However, irrespective of the type of
a stimulus and of the genotype, extrusion of 2 PB and formation of haploid
female pronucleus, i.e. reaction typical for fertilization, always predominates.
The initial ploidy of the parthenogenetic embryo can be determined on the
basis of the number of pronuclei and polar bodies. Eggs which extruded 2 PB
and formed one pronucleus (the reaction occurring in the majority of activated
eggs) and eggs which underwent immediate cleavage were classified as potentially haploid. Eggs which did not extrude 2 PB and had one pronucleus, two
pronuclei or more than two nuclei were qualified as potentially diploid. Among
these eggs those with two pronuclei were most numerous (Table 2). Eggs with
a single diploid pronucleus were observed only occasionally. Thus electric
stimulation does not produce similar effects in mouse eggs to cooling in
rabbit eggs (Chang, 1954).
Preimplantation parthenogenetic development
541
Eggs with two pronuclei were classified as potentially diploid on the assumption that the two nuclei contribute chromosomes to the metaphase plate of the
first cleavage thus restoring diploidy. Graham (1971) observed that binucleated
eggs developing in vitro divide eventually into two cells (a form of delayed immediate cleavage), each with one pronucleus; these eggs become therefore secondarily haploid. Such eggs should undergo first cleavage at a normal time schedule
and on the 2nd day of development should be composed of four cells. These
observations raised the question of whether eggs stimulated with electric current
and developing in vivo behave similarly or not. If such a phenomenon actually
occurred, then the frequency of 4-cell eggs on the 2nd day should correspond
to the total frequency of eggs which underwent immediate cleavage directly
after activation and of eggs containing two pronuclei. In the group of eggs
obtained from induced ovulations which were sufficiently numerous to carry
out such computation, binucleated eggs amounted to 18-6 % of activated eggs
(Tarkowski et al. 1970), yet the incidence of 4-cells eggs on the 2nd day was low
and equal to the incidence of eggs undergoing immediate cleavage immediately
after the shock (about 2 % - table \{a) in Tarkowski et al. (1970) and Table 4
in the present paper). It seems therefore that in vivo eggs with two pronuclei do
develop into diploid embryos and do not behave as in Graham's experiments
carried out in vitro. This conclusion finds also confirmation in the fact that one
third of 2-cell eggs did not possess second polar body (Table 4) and this is
approximately the percentage of one-cell eggs without 2 PB recovered few
hours after activation.
On the basis of his observation on the behaviour of eggs with two pronuclei,
Graham (1971) suggests that diploid parthenogenetic embryos are formed
exclusively from originally haploid eggs as a result of doubling of the haploid
set of chromosomes. If this process occurred at first cleavage then on the
2nd day one should encounter one-cell eggs with 2 PB and one or two nuclei.
Among 62 eggs collected on the 2nd day there were only four one-cell eggs
(Tables 3, 4). Two of them had 2 PB - one contained two nuclei and the other
one nucleus. From these data it can be inferred that in vivo diploid embryos
arise rarely, if ever, from haploid eggs as a result of suppression of the first
cleavage.
Karyokinesis without cytokinesis resulting in the formation of binucleated
cells was observed starting with the second cleavage. This process may occur
in both cells or just in one cell of 2-cell eggs; 4-cell eggs with one binucleated
blastomere have been also observed. It seems very likely that during next
cleavage both nuclei contribute chromosomes to one metaphase plate which
leads to diploidization of the blastomere. This process can occur independently
in different blastomeres and at various stages and it results in the formation of
haploid-diploid mosaic embryos. The diploid state - either original or produced later by doubling of the haploid set - does not always prevent further
doubling of the number of chromosomes. Such a possibility is exemplified by
542
A. WITKOWSKA
an embryo containing a tetraploid metaphase plate. One gets the impression
that disturbances in cytokinesis are peculiar to parthenogenetic origin of embryos
rather than to haploidy itself. It is interesting that during early cleavage binucleated blastomeres were observed only in eggs from induced ovulations.
Among embryos developing from spontaneously ovulating eggs n/2n mosaics
were encountered for the first time on the fifth day. Suppression of cell divisions
must be therefore a general feature of parthenogenetic embryos but in those
originating from spontaneous ovulations it does not occur until late cleavage
stages.
On the 1st day of development haploid eggs decidedly predominate over
diploid eggs irrespective of the strength (voltage) of the applied stimulus (Table 2).
On the 4th and 5th day haploids still prevail but to a smaller degree (Table 7),
because some of them become mosaics. It is very likely that the number of
mosaic embryos was actually larger as ploidy was often determined on the basis
of only one or two metaphase plates. Considerable differences in the size of
nuclei observed in some embryos also suggested mosaicism (Fig. 20). However,
because of variation in the size of nuclei observed also in normal embryos,
attempts to detect mosaicism on this basis were eventually abandoned.
Nearly all eggs activated by electric stimulus undergo at least two cleavages
(cf. percentages of activated and developing eggs on the 1st, 2nd and 3rd day Tables 3 and 4). In general cleavage of parthenogenetic embryos looks normal
(Figs. 8-12), but some eggs contain in the perivitelline space fragments of cytoplasm. Because of the extrusion of a certain amount of cytoplasm, blastomeres
in such eggs are below normal size characteristic for a given stage but do not
differ in size between each other. Extrusion of cytoplasmic fragments occurs
probably during first cleavage as pronucleate eggs examined up to 7 h after
activation do not contain them. One wonders whether this process does not
reflect abnormal cortical reaction following artificial activation.
During the first 3 days of development the cleavage rate of the majority of
parthenogenetic embryos is normal or nearly normal. On the 4th day retardation becomes evident. This is expressed by persistence of cleaving eggs, subnormal mean number of cells in morulae (17-3 as compared to 23-8 - in control
embryos - Table 7) and the almost complete absence of blastocysts. The mean
number of nuclei in embryos recovered on the 5th day was, however, considerably higher than the mean number of nuclei in normal embryos 1 day
younger (Table 7). The delay in the development of parthenogenetic embryos
measured in terms of the number of cell cycles is therefore less than one day.
Mitotic activity on the 5th day continues to be high. During 24 h under consideration the frequency of blastocysts increased from 3-5 % to 50 %. However,
many embryos have not undergone transformation into blastocysts despite
accumulation of a very large number of cells, considerably exceeding the
number of cells at which cavitation normally begins (Fig. 24, Tables 5-7). The
ability to secrete blastocoelic fluid by the cells of parthenogenetic embryos
Preimplantation
parthenogenetic
development
543
seems to be more impaired than cell divisions. The incidence of haploid, mosaic
and diploid embryos among morulae and blastocysts is similar, which would
suggest that transformation of morulae into blastocysts is not connected with
ploidy. At the same time in each of the three groups - n, njln, 2n - blastocysts
contain on the average more cells than morulae (Table 8).
Embryos recovered on the 5th day display clear correlation between the
degree of ploidy and the number of nuclei (Table 8; Fig. 24). Haploid embryos
were built of the greatest number of cells, diploid embryos of the smallest,
mosaic embryos being intermediate. There are two possible explanations of
these observations: (1) actual difference in the duration of cell cycle in n and In
blastomeres, and (2) diploidization by suppression of cytokinesis followed at
the next cleavage by fusion of two haploid nuclei which does not increase the
number of nuclei for one cycle.
Generally speaking eggs from spontaneous ovulation developed better than
those from induced ovulation. It is difficult to say whether this was due to the
properties of eggs themselves or to the environmental (oviducal) factors, because
only spontaneously ovulating females were pseudopregnant. The second possibility should be taken into account in view of the observations by Whittingham
(1968), according to whom development of fertilized eggs in explanted oviducts
depends to some extent on the phase of the oestrus cycle of the female from
which the oviducts have been taken.
On the first day of development no morphological differences were found
between eggs from spontaneous ovulation and those from induced ovulation.
Differences appeared on later days and were expressed by more frequent
occurrence of binucleated blastomeres and cytoplasmic fragments in eggs from
induced ovulations. In superovulated females a sudden decrease in the number
of developing eggs was observed between the third and the fourth day (a drop
from 40 % to 17 %, Tables 4, 6), while in spontaneously ovulating pseudopregnant females the percentage of developing eggs did not drop below 50 %
of recovered eggs even on the fifth day of development. Comparison of these
figures suggests inferior viability of eggs from induced ovulation. The eggs from
induced ovulations also showed greater tendency towards fragmentation. A few
fragmenting eggs were observed as early as a few hours after activation; although
these eggs might have been from an earlier ovulation induced by PMS itself
(Stern & Schuetz, 1970), it does not seem to be so, because control oviducts did
not contain on the first day fragmenting eggs. A marked increase in the number
of fragmenting eggs occurs on the second, and especially on the third day when
they amount to 33 % of all eggs (Table 4). In spontaneously ovulating pseudopregnant females only 7-5 % of eggs recovered on the 3rd day were fragmenting.
Eggs in control oviducts behaved similarly: after induced ovulation fragmentation began earlier (on the second day) and was more intense (Tables 3, 4). In
these females degenerated eggs also appeared earlier than in spontaneous
ovulators. These data are in accord with observations of McLaren & Ward
544
A. WITKOWSKA
Orsini (1968), according to whom fragmentation and degeneration of eggs occur
earlier in cyclic females than in pseudopregnant ones.
The fate of parthenogenetic embryos at the time of implantation and after
implantation is described in the second part of this study (Witkowska, 1973).
I wish to express my thanks to Professor Dr A. K. Tarkowski for his helpful advice and for
his invaluable comments on the manuscript.
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{Received 4 January 1973, revised 11 April 1973)