/. Embryo!, exp. Morpli. Vol. 30, 3, pp. 547-560, 1973
547
Printed in Great Britain
Parthenogenetic development of mouse embryos
in vivo
II. Postimplantation development
By ANNA WITKOWSKA 1
From the Department of Embryology, Zoological Institute,
University of Warsaw
SUMMARY
CBA-p and CBA-T6T6 females were mated with vasectomized males of A strain and early
in the morning the eggs were activated in situ with the electric shock of 30, 40 and 50 V.
Females were killed between the 5th and the 10th day of pregnancy and the implantation
sites were studied histologically or their content was examined under the dissecting microscope.
Of the uterine horns, 43-6 % contained at least one implantation and the mean number of implantations per horn was 0-76. Altogether 152 implantations were collected. The implantation rate was twice as high in older females (12 weeks and over) as in young ones (6-8
weeks). The number of living embryos decreased with every day so that on the 9th and 10th
day only 2 out of 86 embryos were alive (2-3 %). With one exception all embryos which were
alive at the time of examination were retarded in development for approximately 1 day. The
most advanced embryo was at the 8-somite stage. Two attempts aimed at increasing the
synchrony between the embryos and the uterus at the time of implantation (activation
immediately after delayed mating and transfer of 4-5-day embryos to 3-5-day uterus) did not
improve the survival of embryos after implantation.
INTRODUCTION
Preimplantation development of mouse eggs activated in situ with an
electric current was described in Part I of this study (Witkowska, 1973). This
part deals with implantation and postimplantation development of parthenogenetic embryos up to the 10th day of pregnancy. Preliminary results of these
experiments were previously reported (Tarkowski, Witkowska & Nowicka,
1970).
Development of parthenogenetic rabbit embryos till term was claimed as long
as thirty years ago by Pincus (1939 a, b) and Pincus & Shapiro (1940), but up to
now these results have not been repeated. The most advanced parthenogenetic
rabbit embryos obtained by Thibault (1949) and Chang (1954) were unimplanted
blastocysts. Neither Thibault nor Chang found any implantations in females
autopsied between the 7th and 18th day of pregnancy. In a recent review of
1
Author's address: Department of Embryology, Zoological Institute, University of
Warsaw, 00-927/1 Warsaw, Poland.
548
A. WITKOWSKA
parthenogenesis in vertebrates, Beatty (1967) expresses the view that the unquestionable evidence for the birth of a parthenogenetic mammal is still lacking
but the results obtained so far do not exclude such a possibility.
Although the present study proves that parthenogenetic embryos of the
mouse can occasionally develop as far as to mid-term, the chance of development of a parthenogenone till term appears at present very small.
MATERIALS AND METHODS
Spontaneously ovulating females of CBA-p and CBA-T6T6 inbred strains
mated with vasectomized males of A strain were used for experiments. The
operations were carried out on the day of vaginal plug (= 1st day) between
8 and 10 a.m. Eggs were stimulated with an electric current in situ, according to
the technique previously described (Tarkowski et al. 1970).
Females were autopsied between the 5th and 10th day of pregnancy. Whole
uterine horns (5th day) or implantation swellings alone (6-9th days) were fixed
in Heidenhain's fixative (Susa), sectioned at 6//m and stained with Ehrlich's
haematoxylin and eosin. The majority of implantations from the 9th day and all
from the 10th day were torn open and the content was examined under the
dissecting microscope.
In order to prolong the preimplantation development of activated eggs, in
a number of females the oviducts were ligated at the tubo-uterine junction
immediately after applying the electric shock. After 4 days the oviducts were
rinsed and the recovered morulae and blastocysts were transplanted to the uterus
of the 3-5-day pseudopregnant females of A, CBA-p or CBA-T6T6 strains
(modified technique of McLaren & Michie, 1956).
RESULTS
The results of all experiments are summarized in Table 1. All calculations
refer to a uterine horn rather than to a female, because animals were operated
either unilaterally or bilaterally. The material has not been divided according
to the strain of females (CBA-p versus CBA-T6T6) because no differences were
found in the results. No clear differences were observed either as regards the
voltage of the shock (30, 40 or 50 V). The mean number of implantations per
horn and the survival rate of embryos were similar in all the three groups.
The implantation rate is correlated with body weight and/or age of females
(Table 2). The number of implantations per horn increases markedly in the
group of mice weighing at least 21 g and in the group of mice over 12 weeks
old. Weight and age are undoubtedly correlated with each other. Older females
probably ovulate more eggs than young animals and are generally better breeders.
In Table 3 the material is arranged according to the day of autopsy and the
way of examination (histology versus dissection). Only embryos showing clear
Postimplantation
parthenogenetic
549
development
Table 1. Implantation of parthenogenetic embryos in
various experimental groups
No. of
females
No. of
uterine
horns
Mating at night
23
46
Delayed mating
14
28
37
74
40
28
38
50
31
62
Total
96
174
Voltage
30
Total
No. of
Mean no. of
horns with No. of
implantaimplanta- implanta- tions per
tions
horn
tions
22
(47-6 %)
12
(42-8 %)
34
(460 %)
41
0-89
19
0-68
60
0 81
14
(36-6%)
28
(45-1 %)
76
(43-6 %)
30
0-80
42
0-68
132
0-76
Table 2. Relationship between the number of implantations and the
weight and age of females
Weight (g)
15-17-5 18-20-5 21-23-5
No. of females
No. of experimental horns
No. of horns with implantations
No. of implantations
Mean no. of implantations per
horn
Age (weeks)
6-8
9-11
12
26
36
20
34
19
24
47
63
36
59
33
48
15
24
25
17
14
31
(31-9 %) (380 %) (69-4 %) (288 %) (424 %) (64-6 %)
22
43
46
32
17
54
0-47
0-68
1 26
0-54
0-51
112
signs of necrosis were classified as dead. On the 5th, 6th and 7th day it is often
difficult to make such a distinction and it is quite possible that some embryos
classified as alive were in fact dead at the moment of examination. Starting with
the 8th day, living embryos can be distinguished from dead ones with much
greater certainty.
As shown in Part I (Witkowska, 1973), the rate of development of parthenogenetic embryos slows down in the second half of preimplantation period. Consequently they may not be ready for implantation at the moment of the highest
receptivity of the uterus. With this fact in mind two attempts were undertaken
to increase synchronization between the embryos and the uterus.
No. of
uterine
horns
8
16
12
49
83
19
187
Day of
development
5th
6th
7th
8th
9th
10th
Total
5
(62-5 %)
6
(37-5 %)
6
(500 %)
17
(34-7 %)
42
(50-6 %)
8
(42-1 %)
84
(44-9 %)
73
16
14
8
—
3
0
—
24
17
8
—
52
100
2
1
1
98
15
72
10
1
—
—
Resorptions
152
16
81
27
10
9
9
No. of
implantations
* These figures include data published previously (Tarkowski et al. 1970).
28
1
4
5
9
0
0
—
2
7
9
10
—
—
0
—
Embryos
9
No. of
implantations
]Dissection
9
Histological examination
No. of
horns with No. of
Resorpimplanta- implantations
tions
Embryos
tions
26
7
(77-7 %)
5
(500 %)
3
(16-6%)
1
(1-3%)
1
(8-3 %)
9
Embryos
Total*
126
15
80
24
5
2
0
Resorptions
Table 3. Development of parthenogenetic embryos up to 10th day of pregnancy {combined data from Tables 1 and 4),
all voltages combined {30, 40, 50 V)
0-8
0-8
0-9
0-5
0-8
0-6
11
Mean
no. of
implantations
per horn
>
VI
u
ITK<
Postimplantation parthenogenetic development
551
Table 4. Transplantation of parthenogenetic morulae and
blastocysts to pseudopregnant recipients
Embryos transferred
Total no.
Successful
Transfers
Total
no.
Morulae
Blastocysts
13
43
25
18
8
(61-5%)
29
16
13
Implantations
46-5 %
20
700%
No. of
females and
day of
autopsy
6x8
6x9
1x10
(1) Females were mated with vasectomized males early in the morning and
activation was carried out immediately after the copulation. This procedure did
not improve the results - neither were more embryos implanted nor did they
develop better (Table 1).
(2) Embryos 4-5 days old (morulae and blastocysts) recovered from locked
oviducts were transplanted to the uterus of recipients in the 4th day of pseudopregnancy (Table 4). Although the percentage of implanting embryos was high
(46-5 %), the survival rate after implantation was not improved. Implantations
were examined on the 8th, 9th and 10th day but not a single living embryo was
found.
Description of embryos
Fifth day
The uteri of four bilaterally operated females (activation at 30 V) contained
9 embryos: 6 blastocysts, 1 morula and 2 irregular forms.
The blastocysts displayed great variation as regards morphology and size
of the blastocoel. In the best-developed blastocyst (Fig. 1), which closely adhered
to the uterine epithelium, entoderm was already present underneath the inner
cell mass and the trophoblastic cells on the abembryonal pole began to transform into giant cells; in the inner cell mass there were numerous mitoses. Fig. 2
shows an early but otherwise normally built blastocyst. One of the blastocysts
had an inner cell mass composed of a few cells only and resembled a trophoblastic vesicle (Fig. 3).
A 36-cell morula exemplifies multicellular embryos which do not undergo
transformation into blastocysts (Fig. 4). Such morulae are encountered relatively often among 4-5-day unimplanted embryos (Witkowska, 1973). One of the
two irregular embryos is shown in Fig. 5. It was composed of two groups of
cells loosely attached to each other; some cells were binucleated.
As can be judged from the presence of 'imprints' on the walls of crypts all
embryos must have adhered closely to the uterine epithelium and detached
themselves secondarily as a result of fixation.
E M B 3O
A. WITKOWSKA
Postimplantation parthenogenetic development
553
Sixth day
Out of nine embryos eight were classified as alive. As regards morphology
and developmental stage the embryos vary from an irregular group of vacuolated
cells of trophoblastic character (Fig. 15) through blastocysts to an advanced eggcylinder. The blastocysts were normal in structure (Figs. 10, 11) but retarded
in development for approximately one day. An early egg-cylinder is shown in
Fig. 12. In the most advanced egg-cylinder (Fig. 13) the ectoderm, proximal and
distal entoderm and rudiment of the ectoplacental cone were well developed;
the Reichert membrane and the giant cells were also present. This embryo was
the only one developing at a normal rate and corresponding in size to control
embryos (cf. normal CBA embryos from the sixth day - Figs. 8 and 9).
in the majority of cases the uterine epithelium around the implanting embryos
was completely destroyed or at least partly damaged. The exception was a
shrunken blastocyst (Fig. 14) and a group of vacuolated cells (Fig. 15) which
adhered to the intact epithelium. Decidual reaction was clearly visible around
all embryos including the group of vacuolated cells (Figs. 15, 16).
Seventh day
Out of ten embryos recovered on the 7th day five were at the egg-cylinder
stage (Figs. 18-21). All were classified as alive but two of them were most
probably dying. As regards the size all parthenogenetic embryos were much
below the controls (cf. normal CBA embryo from the 7th d a y - Fig. 17). The
trophoblast of parthenogenetic embryos underwent normal transformation
FIGURES 1-7
Figs. 1-5 represent parthenogenetic embryos obtained on the
fifth day of development.
Fig. 1. Normally built blastocyst showing the entoderm underlining the inner cell
mass. On the abembryonic pole one trophoblastic cell has undergone transformation
into a giant cell. Uterine epithelium is intact, x 300.
Fig. 2. Normally built blastocyst with clearly visible inner cell mass and a small
blastocoele. The imprint on the uterine epithelium proves that originally the
blastocyst adhered to the walls of the crypt, x 300.
Fig. 3. Blastocyst resembling a trophoblastic vesicle: the inner cell mass is built of
only a few cells and is not seen on this section, x 300.
Fig. 4. Normal-looking morula. Although it is composed of 36 cells, it has not been
transformed into a blastocyst. x 300.
Fig. 5. Irregular embryo composed of two groups of cells varying in size. Two
nuclei are seen in one cell, x 750.
Figs. 6, 7. Uterine horns with implantations, x 2.
Fig. 6. Seventh day. Implantation marked with an arrow is seen in Fig. 20.
Fig. 7. Ninth day.
36-2
554
A. WITKOWSKA
16
Postimplantation
parthenogenetic
development
555
into giant cells (Fig. 22). Among the resorbed embryos 3 must have died quite
recently as the outlines of the egg-cylinder were still visible.
Eighth day
Out of 16 implantation swellings only three contained living embryo. The
embryos were well-formed egg-cylinders (Figs. 23, 24) comparable in size and
developmental stage to 1-day-younger control embryos (cf. Fig. 17). Other
implantation swellings contained small degenerated egg-cylinders or structureless mass of dead cells.
Ninth day
Out of 62 implantations collected, eight were examined histologically and
all prove to contain dead embryos. In six implantation swellings the dead
embryos were still recognizable - all must have died at the stage of an advanced
egg-cylinder exemplified by 1-day-younger parthenogenetic embryos shown in
Figs. 23 and 24.
Among 54 implantation swellings which were dissected one contained a
living egg-cylinder (Fig. 26). It measured about 700 /im in length and was
composed of embryonic and extra-embryonic parts and a well-developed
ectoplacental cone. The ploidy of this embryo was not determined.
FIGURES
8-16
Embryos in Figs. 8-16 are 6-day-old (Figs. 8, 9, normal embryos,
Figs. 10-16, parthenogenetic embryos). All figures except Fig. 16, enlarged
300 times.
Figs. 8, 9. Two normal embryos from a CBA female exemplifying
variation in size and developmental stage between sister embryos.
Fig. 8. Early egg-cylinder.
Fig. 9. More advanced egg-cylinder composed of embryonic and extraembryonic
parts.
Fig. 10. Regular blastocyst. Although it adheres to the walls of the crypt, the
epithelium remained intact.
Fig. 11. Blastocyst with secondarily obliterated blastocoel. The uterine epithelium
has only been damaged by the trophoblast over a very small area.
Fig. 12. Abnormal early egg-cylinder. The uterine epithelium has been destroyed
around the embryo.
Fig. 13. Embryo at the stage of early egg-cylinder: a layer of proximal entoderm,
single cells of distal entoderm, Reichert membrane and the rudiment of ectoplacental
cone are present. Trophoblast is in direct contact with the uterine mucosa. This
embryo resembles normal embryos as regards morphology and size (cf. Figs. 8, 9).
Fig. 14. A very small blastocyst with a small cavity. Uterine epithelium intact.
Figs. 15, 16. A group of vacuolated cells adhering to the epithelium. Although the
epithelium has not been damaged, decidual reaction in the uterine mucosa has
begun and is seen around the crypt. Fig. 15, x 300, Fig. 16, x40.
556
A. WITKOWSKA
26
Postimplantation parthenogenetic development
557
Tenth day
Out of 12 implantation swellings only one contained a conceptus. It was
a morphologically normal 8-somite embryo. The head folds were still open and
the heart did not beat. The amnion and allantois were normally developed.
The embryo was destroyed prior to karyological examination.
DISCUSSION
Before implantation (4th and 5th day of pregnancy) there are on the average
1-5 morulae and/or blastocysts per one uterine horn (Witkowska, 1973). The
mean number of implantations per horn is 0-76 (Table 1). From the comparison
of these two figures it follows that about a half of morulae and blastocysts do
not implant. Despite this selection the mortality rate continues to be high after
implantation - with every day the proportion of dead embryos increases
(Table 3). Tt appears that there are two periods of high mortality of parthenogenetic embryos: at implantation and at the stage of an advanced egg-cylinder.
The embryos which die at implantation or shortly thereafter include (1) irregular forms (Figs. 5, 15), (2) multicellular morulae which have not cavitated
(Fig. 4), and (3) morphologically normal blastocysts which did not succeed
in destroying uterine epithelium (Figs. 10, 14). In all the above cases the
uterine epithelium remained intact up to the 6th day, which precluded any
further development. It is interesting to note that the decidual reaction was
present around each embryo, including those clearly irregular. This proves
that the decidual changes in the mucosa and the destruction of the uterine
epithelium are brought about independently by two different factors and that
the first process precedes the latter. This conclusion is in agreement with
FIGURES
17-26
Fig. 17. Normal 7-day-old embryo of a CBA strain. x200.
Figs. 18-21. Parthenogenetic 7-day-old embryos at the stage of egg-cylinder.
Although trophoblast is in contact with the mucosa, development has not proceeded beyond the stage characteristic for the 6th day. Signs of degeneration are
already visible, x 200.
Fig. 22. Trophoblastic giant cells from a 7-day-old parthenogenetic embryo, shown
in Fig. 21. x750.
Fig. 23. Normally built 8-day-old egg-cylinder with a narrow proamnion cavity;
embryonic and extra-embryonic parts are clearly separated. This embryo corresponds to normal embryos one day younger, x 200.
Fig. 24. Another 8-day-old embryo at the stage of an advanced egg-cylinder,
delayed in development for 1 day. x 200.
Fig. 25. Dead embryo from a 9-day implantation. Death must have occurred at
a stage represented by the embryo shown in Fig. 23. Both the egg-cylinder itself
and the Reichert membrane lined with entoderm are still visible, x 200.
Fig. 26. Nine-day-old embryo dissected from the uterus. Description in the text.
ca. x200.
558
A. WITKOWSKA
observations made by Tarkowski (1962) on interspecific implantation of rat
and mouse eggs and with the results of descriptive and experimental studies on
implantation in the rat (Blandau, 1949).
It is known that in the mouse decidual changes cannot be induced by beads
(glass or acrylic polimer MG) and unfertilized eggs, the presence of a blastocyst being indispensable (McLaren, 1968; McLaren & Ward Orsini, 1968). The
essential role in this process is played by trophoblast rather than inner cell mass
(Gardner, 1971). In this context it is worth recalling that decidual reaction was
observed around an irregular group of cells (Figs. 15, 16). These cells were
highly vacuolated and must have attained the physiological properties of trophoblastic cells. Presence of decidual reaction in all implantation crypts proves
in addition that all parthenogenetic embryos discovered on histological preparations must have been alive at the time of implantation.
The observations made on the implanting parthenogenetic embryos also
provide information regarding the mechanism of the loss of the zona pellucida
in vivo. Some of the authors studying this problem (Cole, 1967, in the mouse;
Dickmann &Noyes, 1961, and Dickmann, 1967, in the rat) are of the opinion
that the same mechanism operates in vivo as in vitro, i.e. embryos actively
escape ('hatch') from the zona through the cracks formed as a result of high
internal pressure. However, Potts & Wilson (1967) have shown that in utero
the zona pellucida undergoes progressive vacuolation and dissolution at the
implantation site. The work of McLaren (1970) and of Mintz (1971) provides
clear evidence that in normal pregnancy the uterus secretes lysins which dissolve
the zona pellucida. The presence among the implanting parthenogenetic embryos of zona-free morulae and irregular forms also demonstrates that the loss
of the zona does not require mechanical action (pressure on the part of the
embryo) and that lytic factors must be involved.
Many of the parthenogenetic embryos, despite normal structure, have a
limited ability to destroy uterine epithelium and consequently enter into
direct contact with mucosa over a limited area only. This may be one of the
reasons for the delayed rate of development of some of the embryos and their
death on the 6th and 7th day at the stage of an early egg-cylinder (Figs. 18-21).
These observations confirm Tarkowski's conclusion (1962) that the establishment of a direct contact with the uterine mucosa is a prerequisite of progressive
changes in the inner cell mass.
The next period of increased mortality rate of parthenogenetic embryos
occurs at the stage of an advanced egg-cylinder, i.e. at a stage characteristic for
the 7th day of normal development. The embryos dying at this stage had already
developed large ectoplacental cone and conspicuous Reichert membrane with
distal entoderm on one side and giant cells on the other. No characteristic syndrome of developmental anomalies was observed, but starting with the 7th day
all embryos showed retardation in development amounting to 1 day. Two living
embryos found on the 9th and 10th day however provide evidence that de-
Postimplantation parthenogenetic development
559
velopment of parthenogenetic embryos can occasionally proceed beyond this
stage.
In Part I (Witkowska, 1973) of this study it was shown that parthenogenetic
morulae and blastocysts include haploids, diploids and n/2n mosaics. As mosaics
develop from eggs originally haploid, the incidence of haploids among implanting embryos is lower than at the very beginning of development, but
still amounts to 60 % (cf. Part I, Tables 2, 7 and 8). Up to implantation the
development of parthenogenones does not appear to depend on the degree
of ploidy. Probably this is no longer true after implantation. In all other vertebrates studied in this respect haploidy is usually lethal during embryogenesis
(cf. Beatty, 1967) the only known case of survival till adulthood being that of
haploid frogs produced by Miyada (1960). In mammals survival of spontaneous
haploid embryos beyond implantation has never been observed. The present
experiments did not provide any information regarding the ploidy of implanted
embryos - neither the degenerating ones nor those which survived to the day
of examination. By inference from what is known about the effect of haploidy
on embryonic development in inframammalian vertebrates it can be postulated
that the high mortality occurring soon after implantation is mainly caused by
haploidy. The fate of n/2n mosaics may vary depending on the proportion of
haploid and diploid cells in the given embryo. Assuming that haploid embryos
are eliminated shortly after implantation and that only diploidy ensures further
development, one could expect that the ratio between resorptions and embryos
would approximately correspond to the ratio of haploid (including n/2n
mosaics) to diploid embryos, as observed before implantation. The observations
did not confirm this expectation. On the 9th and 10th day of pregnancy only
two living embryos were found among 86 implantations (2-3 %). It follows
that the majority of diploid embryos die as well before the 9th day.
Graham (1971) put forward the hypothesis that death of diploid parthenogenetic embryos is a result of extensive homozygosity which exposes recessive
lethal genes. This hypothesis is based on the assumption that diploid embryos
must develop from originally haploid embryos by doubling the number of
chromosomes, and not from binucleated 1-cell eggs (potentially diploid) because
the latter-according to Graham - become secondarily haploid as a result of
delayed immediate cleavage. However, the data presented in Part I and the
observations on the development of electrically stimulated eggs in vitro (unpublished observations) show that, first, haploid eggs rarely, if ever, regulate
to diploidy by suppression of the first cleavage, and, second, contrary to the
observations made by Graham, 1-cell eggs with two pronuclei can develop into
diploid embryos. These embryos would not be fully homozygous as they carry
two haploid sets of chromosomes produced at second meiotic division. On the
other hand there is no doubt that diploid cells in n\2n mosaics are fully homozygous. Taking into account the fact that the present experiments were carried out
on eggs of inbred strains one can hardly assume that the expression of lethal genes
560
A. WITKOWSKA
might be the main, or at least the only reason of the death of diploid parthenogenones. The significance of this factor should not be, however, underestimated.
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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