/. Embryol. exp. Morph. Vol. 41, pp. 79-92, 1977
Printed in Great Britain © Company of Biologists Limited 1977
79
Factors affecting the time of formation of
the mouse blastocoele
ByROSITA SMITH 1 AND ANNE McLAREN 2
From the M.R.C. Mammalian Development Unit,
University College, London
SUMMARY
In normal mouse embryos developing in vivo, the first appearance of the blastocyst cavity
was found to be associated more closely with developmental age, judged by cell number, than
with chronological age, i.e. elapsed time since ovulation. When development was slowed by
in vitro culture, formation of the blastocoele was delayed. However, cell number itself was
not a critical factor, since the number of cells per embryo could be doubled or tripled or
halved by experimental manipulation without substantially affecting the timing of blastocoele
formation. Experiments in which one cell division was suppressed with cytochalasin-B,
leading to tetraploidy, showed that the number of cell divisions since fertilization was also
not critical. A possible role is suggested either for nucleocytoplasmic ratio, or for the number
of nuclear or chromosomal divisions or DNA replications since fertilization, all of which
increase during cleavage.
INTRODUCTION
Morphogenesis in the preimplantation mouse embryo involves (1) about six
cleavage divisions, from the 1-cell stage onwards, (2) the process of compaction
during which the cell surfaces flatten and come into close apposition with one
another, so that outlines of individual blastomeres are no longer easily seen,
(3) the formation of tight junctions around the periphery of the embryo, and (4)
the secretion of intercellular fluid. Compaction, first reported by Lewis &
Wright (1935), starts at the 8-16-cell stage, is dependent on the presence of
calcium (Whitten, 1971), and marks the beginning of tight-junction formation
(Ducibella & Anderson, 1975). Fluid released from cytoplasmic vesicles accumulates in the intercellular spaces, which become confluent and enlarge (Calarco
& Brown, 1969), while the peripheral tight junctions eventually come to constitute an effective permeability seal, the zonula occludens (Ducibella,
Albertini, Anderson & Biggers, 1975). In this way the blastocyst forms, enclosing
a fluid-filled cavity, the blastocoele. Some data on cell number in morulae and
blastocysts of a number of different mouse strains 1\ days post coitum is
given by McLaren & Bowman (1973).
1
Author's address: University of Chile, Faculty of Agriculture, Casilla 1004, Santiago,
Chile.
2
Author's address: M.R.C. Mammalian Development Unit, University College, Wolfson
House, 4, Stephenson Way, London NW1 2HE, U.K.
6-2
80
R. SMITH AND A. MCLAREN
Little is known of the factors determining the time of first appearance of the
blastocoele. The studies of Tarkowski & Wroblewska (1967) on blastocoele
formation after isolation of blastomeres at the 4-cell or 8-cell stage suggested
that cell number was not important. The conclusion sometimes drawn, that
elapsed time since fertilization (i.e. embryo age) is the determining factor, may
not be justified, as variables that increase throughout the preimplantation period
include not only the age of the embryo and the number of cells, but also the
number of cell divisions and chromosome and DNA replications since fertilization, and the nucleo-cytoplasmic ratio.
The present work was designed to distinguish between some of these factors.
The relative importance of age and cell number was assessed in normal embryos,
culture was used to retard development, aggregation and two-cell separation to
increase or reduce cell number, and treatment with cytochalasin-B to suppress
one cell division.
MATERIALS AND METHODS
All the embryos used were derived from spontaneously ovulating females
belonging to the randomly bred Q strain. The mice were kept under controlled
lighting conditions with 16 h light, and 8 h dark centred on either midnight or
2 p.m. Ovulation was assumed to have occurred at the midpoint of the dark
period preceding the finding of the copulation plug.
Embryos were cultured at 36 °C in a gas phase of 10 % CO 2 in air, under
paraffin oil in 20 [A drops of either Brinster's medium (1965), modified according to Bowman & McLaren (1970), or medium 16 (Whittingham, 1971).
Removal of the zona pellucida and aggregation of 2-cell or 8-cell stages was
performed as described in Bowman & McLaren (1970). Separation of single
blastomeres of 2-cell stages (Tarkowski, 1959a, b) was carried out in calciumand magnesium-free Brinster's medium. The time required for separation of the
sister blastomeres varied from 2 to 10 mins depending on the stage of theintermitotic period of the treated eggs. Gently pipetting of the eggs was usually
carried out in order to hasten the separation. Isolated blastomeres were washed
and placed in separate drops of culture medium. Incubation of 2-cell embryos
in the presence of cytochalasin-B (10 /*g/ml) and 1 % dimethyl sulphoxide was
used to suppress second cleavage and induce tetraploidy (Snow, 1973).
Embryos were classed as nascent blastocysts as soon as one or more blastocoelic spaces could be seen under the dissecting microscope (x 50). Initially,
embryos were also examined under a Zeiss RA microscope fitted with Nomarski
interference optics, but this procedure was discontinued as there was seldom
any problem in deciding how an embryo should be classified. For cell-counting,
embryos were air-dried by the technique of Tarkowski (1966), stained with
lactic-acetic orcein and examined with the aid of a camera lucida microscope
attachment.
Blastocoele formation
81
RESULTS
In vivo
Pregnant females were killed at intervals on the 4th day of pregnancy, from
80 to 90 h after ovulation. The embryos were flushed from the uterus, examined
under the dissecting microscope, and classified as morulae or blastocysts.
Those blastocysts in which cavitation was judged to be just beginning ('nascent'
blastocysts) were identified. In each category, a proportion of the embryos were
fixed and air-dried, so that the cells could be counted.
During the ten hours studied, the mean cell number of the embryos increased
from 22-0 to 44-8 (Table 1). At the beginning of the period, only 17 % of the
Table 1. The rate of development of mouse embryos removed from
the uterus on the 4th day of pregnancy
Age
(h after
ovulation)
80
81
82
83
84
85
87
90
No. of
females
No. of
embryos
Estimated
mean cell
number*
Blastocysts
as % of
total
embryos
76
82
102
69
86
78
84
78
220
27-9
31-3
330
339
351
390
44-8
171
31-7
65-7
65-2
66-3
71-8
73-8
80-7
Nascent
blastocysts
as % of
total
blastocysts
61 5
26-9
17-9
22-2
140
8-9
9-4
3-8
* Calculated here and elsewhere as - 2LJIi
—b, where nm and nb are the numbers of
n m +n b
morulae and blastocysts in any age group, and cIU and cb the mean number of cells in morulae
and blastocysts respectively.
embryos were classified as blastocysts, and most of these (61 %) were only just
beginning to cavitate. At the end of the period over 80 % were blastocysts, only
4 % of which had newly cavitated. The '50% blastocyst' point was reached
between 81 and 82 h after ovulation.
In Fig. 1 are plotted the individual cell counts for all the embryos recovered
during this period, whether morulae, nascent blastocysts or other blastocysts.
The peaks around 16 and 32 cells indicate that the cell populations still showed
a marked degree of mitotic synchrony. The transition from morula to blastocyst took place almost entirely between the 28-cell and the 33-cell stage. Of
147 morulae and 232 blastocysts for which cell counts were made, no blastocyst
with less than 24 cells was found, and no morula with more than 34.
In order to see whether the appearance of the blastocoele was more closely
related to the number of cells in the embryo or to its age in hours after ovulation,
82
R. SMITH AND A. MCLAREN
20
r
15
10
1
5
20
25
20
25
30
30
35
40
35
40
45
45
50
50
55
55
I
i n m n
60
65
60
65
Cell number
Fig. 1. The distribution of cell numbers among morulae ( • ) , blastocysts (Q), and
nascent blastocysts (H) (i.e. those just beginning to cavitate) recovered from the
uteri of pregnant mice 80-90 h after ovulation.
we calculated mean cell numbers of the morulae and blastocysts at each age
(Table 2), and mean ages of the morulae and blastocysts at each cell number
(Table 3). For the population as a whole, nascent blastocysts averaged 30-5
cells, with a mean age of 83 hours after ovulation; cell number and age appeared
to be independent of one another. Embryos of a given cell number showed no
consistent difference in mean age according to whether they were morulae or
blastocysts (Table 3); on the other hand blastocysts of a given age always had a
very significantly higher mean cell number than morulae of the same age
(Table 2). This indicates that the time of blastocyst formation was more dependent on cell number or some related variable (e.g. nucleo-cytoplasmic ratio, or
number of cell divisions or DNA replications since fertilization), than on the
age of the embryo measured in hours since ovulation.
In vitro
Embryos were cultured from the 2-cell (40-42 h after ovulation) or 8-cell
(62-64 h after ovulation) to the blastocyst stage, and examined at intervals from
87 to 96 (2-cell) or 81 to 90 (8-cell) h after ovulation. Whether judged by the
percentage of blastocysts or by the mean cell number (Table 4, cf Table 1), the
group cultured from the 2-cell were significantly more retarded than those
cultured from the 8-cell stage, and both were significantly retarded relative to
embryos developed in vivo. However, the rate of cleavage during the period of
examination was similar in the three groups (Fig. 2).
As in vivo, blastocysts contained significantly more cells than morulae at any
83
Blastocoele formation
Table 2. The mean cell number of morulae and blastocysts recovered from the
uterus on the 4th day of pregnancy, classified according to age {in h after
ovulation)
(Here and in Tables, 3, 5 and 6 the number of embryos on which the mean is based
is given in parentheses below the mean±s.E.)
Age (h after
ovulation)
80
81
82
83
84
85
87
90
Morulae
Blastocysts
Nascent blastocysts
19-8 + 1-3
(27)
25-2±l-0
(24)
22-3 ±1-5
(15)
23-6 ±1-5
(16)
26-5 + 1-4
(16)
26-2+1-3
(22)
25-6 + 1-6
(15)
27-8 + 0-9
(12)
32-8 + 2-7
(10)
33-6 + 2 0
(17)
36-0+1-6
(30)
38-0 ±2-2
(21)
37-6±l-8
(32)
38-6±l-4
(38)
43-8 ±1-8
(40)
48-9 ±1-5
(44)
31-7 + 0-6
(6)
30-3 + 1-1
(7)
30-6 + 0-8
(7)
28-8 ±0-8
(6)
30-5 ± 1 0
(6)
30-6 ±1-2
(5)
29-7 ±0-9
(3)
340 ± 0 0
(2)
Table 3. The mean age {in h after ovulation) of morulae and blastocysts recovered
from the uterus on the 4th day of pregnancy, classified according to cell number
Cell no.*
Morulae
Blastocysts
Nascent blastocysts
26-27
84-3 ±0-69
(18)
81-9 ±0-48
(13)
84-4 ± 1 0 8
(8)
84-8 ±0-87
(10)
86-8 ±0-86
(11)
82-5 + 1-96
(10)
84-3 ± 1 0 9
(6)
83-3 ±0-90
(9)
83-7 ±0-63
(12)
82-5 ±0-44
(12)
83-7 ±0-70
(11)
83-9 + 0-72
(17)
83-3 ±0-58
(18)
84-6 ±0-67
(24)
83-0f±0-82
(4)
83-6 ± 1 0 3
(5)
820 ±0-71
(5)
83-5± 1-19
(4)
83-0 + 0-87
(8)
82-3 ±0-84
(6)
83-7±l-18
(10)
28
29
30
31
32
33-34
* Only those cell number categories that include both morulae and blastocysts have been
listed.
f Includes one nascent blastocyst with only 24 cells.
84
R. SMITH AND A. MCLAREN
Table 4. The rate of development of mouse embryos cultured from the
2-cell or 8-cell stage
Cultured from 2-cell
Cultured from i8-cell
Age
(h after
ovulation)
No. of
embryos
Estimated
mean cell
number
Blastocysts
(%)
No. of
embryos
Estimated
mean cell
number
81
84
87
90
93
96
106
112
79
85
53
62
0
0
50
0
22-1
25-1
28-6
32-7
—
—
—
—
8-9
18-8
340
45-2
—
—
840
—
0
0
64
71
54
52
0
45
—
—
21-6
28-4
30-4
37-5
—
—
Blastocysts
(%)
—
—
20-3
33-8
40-7
63-5
—
82-2
given age (Table 5), confirming that the timing of blastocyst formation in normal
embryos is related to cell number. It was therefore not surprising to find that
the mean cell number of nascent blastocysts among cultured embryos (Table 6)
was only slightly below that observed in vivo, though the age at which the embryos
on average achieved the critical number of cells was greater by 2-3 and 5-2 h
respectively for embryos cultured from the 8-cell or 2-cell stage. Removal of
the zona before culture appeared to make little difference to the cell number at
which the blastocyst cavity first appeared (Table 6).
Changes in cell number
To investigate the effect of cell number as such, embryos when they were
first placed into culture were aggregated into doublets or triplets at the 2-cell
or 8-cell stage, or separated into halves (i.e. single blastomeres) at the 2-cell
stage. The controls were embryos cultured without the zona pellucida from the
2-cell or 8-cell stage.
If the time of blastocyst formation is dependent on cell number as such, one
would expect the mean cell number of nascent blastocysts in each of the treated
groups to resemble that of the relevant controls. If on the other hand cell
number as such is not involved, the mean cell number of nascent blastocysts
in the doublet, triplet and single blastomere groups should be respectively twice,
three times and half that of the controls. It is clear from the data given in Table 6
that the second expectation is the more nearly fulfilled. In every case the mean for
the treated group approximates to the double, treble or half value expected.
Evidently neither cell number nor embryo age determines the time of appearance of the blastocyst cavity. Other possible variables that might be involved
include the number of cell or nuclear divisions or DNA replications since
fertilization, and the ratio of nucleus to cytoplasm.
Blastocoele formation
1 65
145
r
Developed
;'/7 vivo
•60
85
40
1-55
35
1-50
30 I
to 1-45
o
1-40
25
1 35
20
1-30
80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
Age (h after ovulation)
Fig. 2. The relation of cell number to age of embryos developing in vivo and
in vitro. Thecalculated regression lines differ significantly from one another in position
but not in slope, x , developed in vivo; • , cultured from 8-cell; O, cultured from
2-celi.
Cytochalasin-B treatment
A further group of embryos was exposed to cytochalasin-B at the 2-cell
stage. This treatment prevents second cleavage, while allowing DNA replication
and nuclear division to proceed normally. After removal from cytochalasin, the
binucleate cells undergo their subsequent cleavage at the normal time. The two
chromosome sets align themselves on a single spindle, resulting in a population
of uniformly tetraploid 4-cell embryos, contemporary with 8-cell stages in the
control group not exposed to cytochalasin. The treated embryos have undergone one fewer cell division than the controls, but the same number of nuclear
divisions and DNA and chromosome replications. Because of the blocked cell
division, the cells are twice as large as controls of the same age, but the nuclear
doubling and resultant tetraploidy mean that the nuclei too may be almost
twice as large. The nucleo-cytoplasmic ratio in an 8-cell cytochalasin-treated
embryo is therefore similar to that in a 16-cell control of similar age.
The rate of development of cytochalasin-treated embryos relative to controls
is illustrated in Fig. 3, and the distribution of cell numbers in treated and
86
R. SMITH AND A. MCLAREN
Table 5. The mean cell number of morulae and blastocysts cultured from the
8-cell or 2-cell stage, according to age {in h after ovulation)
Age
(h after
ovulation)
Cultured from 8-cell
Morulae
Blastocysts
93
21-3 + 0-9
(41)
23-6 + 0-9
(42)
24-3 + 1-3
(20)
27-7 + 0-8
(15)
—
30-4 ±0-7
(5)
31-6±2-l
(15)
37-1 ±2-1
(18)
38-6+1-9
(24)
—
96
—
—
106
—
112
—
61-2 + 4-0
(20)
—
81
84
87
90
Cultured from 2-cell
(
Morulae
Blastocysts
—
—
18-9±l-7
(23)
21-9+1-7
(15)
22-4 + 1-6
(17)
26-1 ±2-3
(9)
—
32O± 1-3
(11)
41-2 ±2-3
(12)
42-0±l-7
(11)
44-1 + 2-1
(16)
—
74-9 ±3-3
(22)
—
control blastocysts in Fig. 4. The mean cell number of treated blastocysts is
slightly less than half that of the controls (23-4 ± 0-7 compared with 52-6 ± 3-2),
as found also by Snow (1973, 1975).
If the time of appearance of the blastocyst cavity depended on the number of
cell divisions since fertilization, the mean cell number of nascent blastocysts
should be similar whether embryos have been treated with cytochalasin or not.
Table 6 shows that the mean is 16-2 in the cytochalasin-treated group, compared
with 28-3 in the appropriate controls. If the relevant factor were the number of
nuclear divisions or DNA or chromosome replications, or the nucleo-cytoplasmic ratio, a two-fold difference would be expected, which is in better agreement
with the data.
DISCUSSION
Blastocoele formation is a compound process, involving both the secretion
of fluid and the formation of tight junctions around the periphery of the embryo.
It is not known whether both sub-processes reach their critical value at the
same time (i.e. shortly before the 32-cell stage in normal embryos), or whether
one requirement is established earlier, so that the timing is related to the
appearance of the other. Junction formation begins at the 8-cell stage, as part
of the process of compaction (Ducibella & Anderson, 1975); the zonula occludens, which forms the actual permeability seal between trophectoderm cells, has
been reported to exclude lanthanum tracer after the 16-cell stage (Ducibella et al.
1975), at 70 h post coitum in Swiss mice. The blastocoele fluid is thought to
Blastocoele formation
87
Table 6. The appearance of the blastocyst cavity in embryos cultured from the
8-cell or 2-cell stage (for 26 and 52 h respectively), with or without the zona pellucida, singly, aggregated in doublets or triplets, separated into halves, or treated
with cytochalasin-B to suppress one cell division
Stage at
start of
culture
8-cell
2-cell
Group
No. of
Blastocysts
embryos
as % o f
cultured total embryos
In zona
180
152 (84-4)
Naked
117
97 (82-9)
Double
80
63 (78-7)
Triple
61
52 (85-2)
In zona
88
70 (79-5)
Naked
66
50 (75-8)
Double
53
41 (77-4)
Triple
58
42 (72-4)
Halff
100
59 (590)
Tetraploid
122
69 (56-6)
Mean cell
no. of nascent
blastocysts*
(mean±s.E.)
Mean age of
nascent
blastocysts
(h after
ovulation)
29-2 + 0-29
(53)
29-2 + 0-27
(43)
52-2 ±0-85
(20)
831 ±1-55
(14)
28-3 ±0-47
(22)
27-3 + 0-52
(22)
52-2 + 0-90
(15)
81-6+118
(14)
12-8 + 0-31
(24)
16-2 + 0-57
(14)
85-3 ±0-37
(69)
84-8 ±0-42
(55)
85-7 ±0-63
(25)
85-9 ±0-41
Q6)
88-2 ±0-33
(32)
891 ±0-58
(28)
89-6 ±0-71
(19)
90-5 + 0-66
(19)
91-5 + 0-50
(28)
95-3±l-36
(23)
* The embryos were inspected at intervals during the culture period. Any nascent blastocysts seen were removed so that their cellss could be counted.
| Cultured for 54 h
be released from cytoplasmic vesicles; these are seen in all preimplantation
stages up to and including morulae, but are rare in blastocysts (Calarco &
Brown, 1969). They are most numerous at the 4-cell stage; subsequently they
decrease in number but increase in size, as though coalescing prior to release.
Calarco & Brown (1969) suggest that vesicle release may begin as early as the
8-cell stage, which would imply that the critical factor in the formation of
a visible blastocoele was establishment of the zonular-j unction permeability
seal.
The earliest embryos in which anti-mouse antiserum was sufficiently excluded
from the interior of the embryo to permit survival of some interior cells (Solter
& Knowles, 1975) proved to contain about 40 cells in toto (McLaren & Smith,
1977). Since this is well beyond the stage at which the blastocoele first appears,
either the zonula occludens is more effective at keeping fluid in the blastocoele
88
R. SMITH AND A. MCLAREN
Time in
culture
2 cell
5-8 Early
Late
Early
Late
cell morula morula blastocyst blastocyst
Li
14 h
22 h
3-4
cell
n fl
I
IkJ
40 h
50 h
62 h
2 cell
3-4
cell
5-8
cell
Early
Late
Early
Late
morula morula blastocyst blastocyst
Fig. 3. A comparison of the rate of development of cytochalasin-treated tetraploid
(white) with control diploid (black) embryos. The height of each block represents
the percentage of embryos that have reached the given stage of development at that
time. After 62 hrs of culture, 180 % of control and 43-5% of cytochalasin-treated
embryos were dead; these have not been taken into account in calculating the
percentages.
than at keeping it out, or a completely impermeable seal is not required for blastocoele formation.
Analysis of the age and cell number of mouse embryos in which the blastocyst cavity was just beginning to form indicated that the timing of the critical
factor was determined by some variable related to cell number rather than to
embryo age. However, experiments in which cell number was either increased or
decreased established that cell number itself was not involved, while suppressing
one cell division with cytochalasin-B showed that the number of cell divisions
since fertilization was also not critical. We therefore conclude that the time of
formation of the blastocoele is determined by some other factor, such as the
number of nuclear divisions or DNA or chromosome replications that have
elapsed since fertilization, or the ratio of nuclear to cytoplasmic material in the
cell.
All these are factors that relate to individual cells, rather than to the embryo as
Blastocoele formation
89
50 r
10 20 30 40 50 60 70 80 90
Cell number
Fig. 4. The distribution of cell numbers among diploid control (hatched) and
cytochalasin-treated tetraploid (white) blastocysts.
a whole. The first three increase in a stepwise fashion: nuclear division coincides
with cell division, chromosome replication occurs somewhat earlier, and DNA
synthesis takes place in mid-cycle. Nucleo-cytoplasmic ratio stays more or
less constant up to the 16-cell stage, increasing approximately four-fold between
the 16-cell and 64-cell stage (Snow, personal communication).
Control embryos, developing in the reproductive tract, showed a mean cell
number for nascent blastocysts of 30-5. This corresponds to a point in cleavage
at which most of the cells have divided five times, the remainder (less than two
on average) have divided only four times, and all have undergone five DNA
replications. For the stepwise variables, this would imply that the initiation of
blastocoele formation requires four nuclear or chromosomal doublings or
five DNA replications in all the cells, or five nuclear or chromosomal doublings
in most of the cells.
The embryos that were retarded by culture, or in which the cell number was
augmented or diminished, initiated blastocoele formation at approximately the
same point in cleavage as the controls, i.e. when all the cells would have undergone five DNA replications and most but not all had divided five times. The
mean cell number was however consistently and significantly less than that
expected from the controls: nascent blastocysts from culture had 5-10%
fewer cells than those that had developed in vivo, doubles and triples fell short
of two or three times the cell numbers of the corresponding cultured controls by
up to 10 %, while halves were again only 94 % of the expected value. In each
case the experimental embryos were subjected to more extensive manipulations
than were the controls: the consistent deficit in cell number would be expected
if each of the experimental treatments led to the death or loss of 5-10 % of the
90
R. SMITH AND A. MCLAREN
blastomeres during the course of the culture period, so that the actual cell number was lower than the theoretical expectation, but the stage at which the blastocoele appears was not affected.
A contrasting picture is shown by the embryos treated with cytochalasin-B.
One cell division is suppressed, and the overall cell number is significantly less
than half the control. Yet the mean cell number of nascent blastocysts is 16-2,
significantly more than half the value for the cultured controls or even the
in vivo embryos. This difference adds some weight to the possibility that the
critical factor may be nucleo-cytoplasmic ratio: the doubling of chromosome
number would not necessarily increase nuclear size by as much as two-fold, so
the nucleo-cytoplasmic ratio corresponding to a control embryo of less than
32 cells might well not be reached in the tetraploids until the 16-cell stage.
Additional evidence exists in favour of the importance of nucleo-cytoplasmic
ratio. In the work of Witkowska (1973) on parthenogenetically activated mouse
eggs, many of the resulting embryos were haploid, with a lower nucleo-cytoplasmic ratio than normal; it was reported that the appearance of the blastocoele was delayed by up to one cleavage division. A similar finding was reported
by Modlinski (1975), who halved nucleo-cytoplasmic ratio by removing one
pronucleus from fertilized eggs.
Our conclusions are consistent with the observations of earlier workers,
that in experimentally manipulated embryos a blastocoele can form when the
number of cells present is very small. Thus Tarkowski & Wroblewska (1967),
isolating single blastomeres from mouse embryos at the 4- and 8-cell stage, reported blastocoele formation in embryos containing as few as four cells (or
even in one case two cells, but there was some doubt in this case as to whether
the fluid-filled cavity was extracellular). Snow (1973) reported blastocoele
formation in embryos containing only two cells, following cytochalasin treatment. Such results have been interpreted by some authors as implying that
blastocoele formation depends on the time elapsed since fertilization. Thus
Tarkowski & Wroblewska (1967) conclude: 'Secretion of the blastocoelic
fluid seems, therefore, to represent a cytoplasmic activity which a cell undertakes after a certain definite period of time and irrespective of the number of
nuclear cycles.' Other authors have echoed this conclusion. Ducibella & Anderson (1975) write '...blastocoele formation, appears to be a developmentally
programmed event scheduled in time but not with respect to cell number
(Tarkowski, 1959)'; Granholm & Brenner (1976) write '...regulation of morphogenetic processes in preimplantation mouse development appears to be less a
matter of blastomere number and more a matter of chronology'.
In a sense the conclusion that the event is scheduled in time is correct: but
our results have shown this time to be measured by a biological, not a chronological, clock. Tarkowski & Wroblewska's 'mini-blastocysts' all underwent at
least four nuclear divisions and hence four chromosome replications; Snow's
two-cell 'blastocysts' were in fact 16-ploid, so again had undergone four
Blastocoele formation
91
chromosome replications. In both cases the embryos may have experienced
five rounds of DNA synthesis, and their nucleo-cytoplasmic ratio may have
resembled that of normal embryos just prior to the fifth cleavage division. In
Snow's cytochalasin-treated embryos, nuclear division was suppressed, suggesting that this factor can be eliminated from the list of possible candidates for
the biological clock.
Tarkowski & Wroblewska (1969) mention that at least one isolated blastomere
from an 8-cell embryo did not divide at all, but nonetheless showed highly
vacuolated cytoplasm. Unless these vacuoles were a sign of degenerative change,
this finding supports the observations of Calarco & Brown (1969), but throws
no new light on the signal for vacuole release, which is the prerequisite for
blastocoele formation.
Although the observations on cytochalasin-treated embryos in this paper, as
well as the earlier findings on haploid embryos, make nucleo-cytoplasmic ratio
an attractive candidate for the biological clock involved in blastocoele formation, one cannot eliminate numbers of chromosome replications or rounds of
DNA synthesis. One possible approach to distinguish between these alternatives
would be to study the time of blastocoele formation in embryos developing
from fertilized eggs cut in two by the technique of Tarkowski & Rossant
(1976). If both pronuclei were left in a reduced body of cytoplasm, an embryo
should be formed in which the nucleo-cytoplasmic ratio was consistently larger
than was appropriate for the number of DNA and chromosome replications;
blastocoele formation should therefore occur precociously if nucleo-cytoplasmic ratio were critical.
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{Received 10 January 1977, revised 30 March 1977)