/ . Embryol. exp. Morph. Vol. 54, pp. 241-261, 1979
Printed in Great Britain © Company of Biologists Limited 1979
241
The effect of prolonged decompaction
on the development of the preimplantation mouse
embryo
By M. H. JOHNSON, 1 J. CHAKRABORTY, 2
A. H. HANDYSIDE 1 , K. WILLISON 3 AND P. STERN 4
From the Department of Anatomy and the MRC Laboratory of
Molecular Biology, Cambridge
SUMMARY
A rabbit antiserum to a mouse embryonal carcinoma cell line blocks compaction of cleaving
mouse embryos. Cell division is not affected up to the 32-cell stage but intracellular junctions
fail to develop. Removal of the antibody at this stage permits compaction to occur and a
normal blastocyst develops. Prolonged decompaction beyond the 32-cell embryo results in
an increasing proportion of malformed blastocysts in which trophectodermal cells predominate
and functional inner cell mass (ICM) cells are reduced or absent. The relationship of compaction to the generation of ICM and trophectoderm lineages in the intact embryo is discussed.
INTRODUCTION
It has been proposed that the position of a cell within the morula affects its
developmental fate (Mintz, 1965; Tarkowski & Wroblewska, 1967). Cells which
occupy the central position(s) on a radial axis through the embryo tend to
contribute to the inner cell mass lineage (ICM) whereas those at peripheral
points on the axis tend to become trophectoderm (Graham & Deussen, 1978;
Hillman, Sherman & Graham, 1972). The cellular response to position, as
assessed by biochemical (Izquierdo, 1977; Handyside & Johnson, 1978), morphological (Ducibella, 1977) and functional (Graham, 1973; Snow, 1973)
criteria first occurs at the early morula stage shortly after the process of compaction. It has, therefore, been suggested that compaction itself is involved in
generating positional cues, perhaps by creating distinctive 'inside' and 'outside'
microenvironments. In apparent conflict with this proposal, it has been demon1
Authors' address: Department of Anatomy, Downing Street, Cambridge, U.K.
Author's Address: Department of Physiology, Medical College of Ohio, Toledo, Ohio,
U.S.A.
3
Author's address: Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor,
N.Y. 11724, U.S.A.
4
Author's address: Department of Immunology, Biomedicum Box 582, 5-751-23 Uppsala,
Sweden.
2
242
M. H. JOHNSON AND OTHERS
strated that the transient inhibition of compaction by the Fab fraction of a
rabbit antiserum to mouse teratocarcinoma (F9) does not apparently lead to
abnormalities in the differentiation of ICM and trophectoderm (Kemler,
Babinet, Eisen & Jacob, 1977). We report here the results of a more prolonged
incubation of embryos in a rabbit antiserum to an undifferentiated embryonal
carcinoma (ec) cell line (L55770 derived from 129J mice) which also causes
decompaction. Although blastocyst differentiation displays a remarkable resilience in the face of prolonged periods of decompaction, distinct effects on
development have been observed.
MATERIALS AND METHODS
(1) Recovery and culture of embryos
CFLP female mice (Anglia Laboratory Animals), 4-5 weeks old, were
superovulated with 5 i.u. PMS and HCG and mated. Embryos were flushed with
phosphate-buffered medium 1 + 10% inactivated foetal calf serum (PB1 +
protein) (Gibco Ltd.) (Whittingham, 1971) from the oviducts of plug-positive
females 46-48 h after the HCG injection. The 2-, 3- and 4-cell embryos recovered
at this time were placed, still in their zonae, in drop culture in medium 16
containing 0-4% bovine serum albumin (BSA) (Whittingham, 1971). Antiserum, with or without absorption, was added to some of the drops. The embryos
were then cultured under oil in a moist atmosphere of 5 % CO2 in air. At intervals
thereafter over the next 72 h, embryos were examined or sampled as described
in the results. Any embryos which failed to divide within 24 h were discarded
(c.a. 20% regardless of culture conditions). Media were changed every 24 h.
Under these culture conditions, in populations of control embryos, cleavage
to the 6- to 8-cell stage occurred by 70 h post-HCG, compaction extended over
the period 69-78 h post-HCG (mid-point of population 74 h), and initiation of
blastoceolic cavity formation extended over the period 88-98 h post-HCG (mid
point of population 93 h). When embryos were to be transferred from medium
containing antiserum to antiserum-free medium, they were first washed for
5 min in an excess volume of PB1+protein. After 72 h some embryos were
washed and placed in drops of medium RPMI+10% inactivated foetal calf
serum, and then cultured for a further 48-72 h under oil in 5 % CO2 in air. In
some cases, the zona pellucida was removed by brief exposure to acid Tyrode's
solution.
(2) Antiserum
Rabbit antiserum was prepared to LS5770 mouse nullipotent ec cells grown
in vitro (gift of Dr J. Jami). The cells were grown under standard conditions
and harvested by trypsinization (Martin & Evans, 1975). The cell suspension was
washed extensively in phosphate-buffered saline (PBS) and 108 cells in PBS were
emulsified in Freund's complete adjuvant (FCA) (1 volume antigen:2 volumes
Role of compaction in blastc cyst formation
243
FCA) and injected into multiple subcutaneous sites of a New Zealand White
rabbit. One month later the animal was boosted with 107 cells intravenously and
bled 10 days later. The separated serum was heat inactivated by incubation for
30 min at 56 °C and stored in small aliquots.
Preliminary tests using indirect immunofluorescence suggested that this
antiserum has a broadly similar pattern of activity to one described previously
(Gooding & Edidin, 1974). After absorption with liver and lymphocytes, the
antiserum no longer reacted with lymphocytes but continued to label fibroblasts and some tumour cell types as well as embryonal carcinoma. Further
absorption of the antiserum with fibroblasts removed the cross-reactive fibroblast-tumour activity but not activity against embryonal carcinoma. All the
activity of this antiserum tested against the immunizing cells could be removed
by absorption with either LS5770 nullipotent ec cells or with OTT5568 ec cells
which do not differentiate in culture under normal conditions. In this work the
antiserum was generally used unabsorbed. In some control experiments the
antiserum was used following absorption with Nulli-SCCl or STO fibroblasts
(Martin & Evans, 1975). Undiluted antiserum was absorbed three times at
1 volume of packed cells: 1 volume antiserum. The absorbed antisera were no
longer active in indirect immunofluorescence tests against Nulli-SCCl or STO
respectively; the STO-absorbed serum retained anti-ec activity. Absorption with
Nulli-SCCl removed all effects on mouse embryos: absorption with STO
fibroblasts was without effect.
Antiserum at 1/40 dilution reacted with 2-cell, 4-cell, 6- to 8-cell precompaction
embryos, postcompaction 8-cell embryos, late morulae and both ICM and
trophectoderm of 3^-day, 4^-day and outgrown blastocysts as judged by complement-mediated cytotoxicity and/or immunofluorescence.
The antiserum was diluted 1/10 in medium 16, and dialysed extensively
against three changes of a 100-fold excess of medium 16 at 4 °C for 24 h. The
diluted and dialysed antiserum was cleared of any precipitate, and further
dilutions were made and tested, in preliminary experiments, on the embryos. A
dilution of 1/40 was found to give consistent and complete decompaction, and
to exert no deleterious or toxic effects on the embryos. Lesser or greater dilutions
of antiserum produced more variable decompaction. Cultures of 2-cell embryos
in 1/40 dilutions progressed through cleavage at the same rate and in the same
proportion (approximately 80%) as control embryos. Thereafter 1/10 diluted
aliquots of 200 jttl were stored at - 7 0 °C and were diluted to 1/40 with 600 p\
of fresh medium prior to use in culture.
(3) Immunosurgery
The modified immunosurgical technique (Handyside, 1978 a) was used. For
all embryos, the zona pellucida was removed as described previously. Embryos
were rinsed briefly, incubated in 1/10 rabbit antiserum to mouse species antigens
for 5 min, rinsed a second time, and then incubated for only 5 min at 37 °C in
244
M. H. JOHNSON AND OTHERS
1/10 guinea-pig complement absorbed on agarose (Cohen & Schlesinger, 1970),
rinsed thoroughly, and incubated in medium PB1 + 1 0 % foetal calf serum for
30 min to permit completion of lysis. After the final incubation, inside cells
were recovered as described previously (Handyside, 1978 a).
(4) Cell counting
For precompaction cleavage stages, visual counting was undertaken. For
later stages, embryos (or parts thereof), were prepared as spreads by the technique of Tarkowski (1966). Any spreads which poorly or excessively dispersed
were not included. The number of mitoses in each spread was recorded.
(5) Electron microscopy
Embryos were fixed for 15 min in 2-5 % glutaraldehyde in 0-1 M-Na cacodylate buffer containing 10% sucrose, rinsed in the buffer containing sucrose,
incubated in 1 % osmium tetroxide in buffer plus sucrose for 15 min, taken
through 5 min rinses in 20%, 70%, 95% and 100% ethanol, infiltrated for
30 min in a 50:50 mixture of propylene oxide and epon. The embryos were
then embedded, singly or in pairs, in epon. Sections were taken at 90-100 jam,
stained with lead (Venable & Coggershall, 1965) and examined on a Phillips
EM 300 electron microscope. For each class of embryo studied, extensive
sections were analysed from a minimum of three embryos.
(6) Embryo transfer
Embryos were transferred ectopically to beneath the kidney capsule of female
mice (Johnson & Dharmawardina, 1972). Seven days later the females were
killed, the sizes of the haemorrhagic nodules recorded, and the segments of
kidneys fixed in 50 % Bouins' solution. After processing, 7 /m\ serial sections were
taken, mounted and stained with haematoxylin and eosin. Uterine transfers
were made to 2^-day pseudopregnant recipients and uteri fixed at 8£ days and
processed as above.
RESULTS
1. Morphology
The morphology of precompaction embryos incubated in antiserum did not
appear to differ from that in controls. However, embryos in antiserum had
failed to compact at 74 h post-HCG, as judged at light microscope level. By
94-96 h post-HCG control embryos either had formed early blastocysts or
consisted of a compact group of cells some of which were undergoing fluid
accumulation prior to blastocyst formation (Fig. 1 and lines 1-3, Table 1). At
the electron microscope level, differences were observed between the inner
presumptive ICM cells and the outer presumptive trophectoderm cells (Fig. Id).
Tight junctional complexes had formed between outer cells which showed
evidence of polarity. Cell flattening appeared in the apical areas of the outer
118-120
94-96
33
69
78
—
—
—
—
—
—
—
—
—
—
—
—
100
—
—
—
—
—
—
—
97-7
Compacted
morulae
Uncompacted
moruiae
67
31
22
—
0-3
100
100
100
—
—
—
—
Blastocysts
—
—
—
—
—
—
—
40
20
3
6
' True'
blastocysts
* C = mediumi without antibody (h incubation).
t A = medium plus antibody (h incubation).
Incubation medium
1. *C(48)
2. fA(24) + C(24)
3. A(36) + C(12)
4. C(24) + A(24)
5. (A(48)
6. C(72)
7. A(24) + C(48)
8. A(36) + C(36)
9. A(48)+C(24)
10. (A(60)+C(J2)
ll.C(24) + A(48)
12. A(72)
Time of
examination
(h post HCG)
—
—
—
—
—
—
—
44
53
36
23
'False'
blastocysts
Embryonic forms expressed as a percentage
—
—
—
—
—
—
—
—
16
27
61
71
'Aggregate'
blastocysts
255
29
27
22
355
168
29
24
223
49
33
73
Total
no. of
embryos
Table 1. Morphology of embryos grown from 2-cell stage (46-48 h post HCG) in medium ±anti ec (1/40)
3
^o
(*^
OJ
3
Ci
246
M. H. JOHNSON AND OTHERS
Fig. 1. Phase-contrast photomicrographs of mouse embryos recovered at the 2cell stage (45-46 h post HCG) and cultured for 48 h in medium 16 + BSA. Note
formation of early blastocoelic cavities, x 345.
presumptive trophectoderm cells facing the zona pellucida. Those areas were
marked by parallel arrays of microtubules (Fig. 2b, c). At this stage of development, microvilli were present at the apical surface of the trophectoderm cells.
Boundaries of ICM cells and lateral contact areas between two neighboring
trophectoderm cells were free of microvilli. Inner cells had formed only a few
gap junctions and were not overtly polarized. In contrast, embryos maintained
in antiserum up to 94-96 h post-HCG consisted of uncompacted masses of cells,
which, in most cases, showed intracellular fluid vacuoles (Fig. 3 and lines 4 and
5, Table 1). At the level of the electron microscope, it was evident that although
cells showed areas of close contact they had failed to form any kind of detectable
specialized junctions and were merely aggregates of morphologically indistinguishable cells. All cells both inside and outside contained vacuolar structures
(Fig. 4).
If embryos incubated in antiserum were washed in control medium by 84 h
post-HCG (lines 7 and 8, Table 1), they were observed to compact within 10 h
(94 h post-HCG) and by 120 h post-HCG had formed blastocysts indistinguishable from the controls. In contrast, incubation of the embryos in antiserum
for longer periods beyond 84 h post-HCG resulted in the increasing incidence of
Role of compaction in blastocyst formation
247
three abnormal types of'blastocysts' (lines 9-12, Table 1). These were classified
as (1) aggregates of fluid-accumulating cells (Fig. 5), which, at the electron
microscope level (Fig. 6 a), were observed to consist of networks of trophoblast-like cells containing vacuolar structures and appearing to form abortive
blastocoelic cavities. Some tight junctional complexes had formed but their
orientation was not clearly tangential as in normal controls. No microtubules
were present near the apical areas of trophectoderm cellular appositions. Long
narrow strips of cytoplasmic processes of two adjoining cells had formed
membrane 'kisses' (Fig. 6 b). (2) 'False' blastocysts (Fig. 7), in which a clear
blastocoelic cavity was present, but which contained either no obvious ICM
or a few fluid-accumulating cells. At the electron microscope level, a few inside
cells could be observed in some embryos, but a large organized ICM was not
evident (Fig. 8). Microvilli were present between adjacent inside cells and between lateral contact areas of trophectoderm. (3) 'True' blastocysts which
appeared to have a normal morphology although often with a reduced ICM.
Electron microscopic analysis confirmed the relatively normal appearance. The
number of embryos examined and their morphology under different conditions
is recorded in Table 1 and summarized in Fig. 9.
2. Cell numbers
Continuous incubation of 2-cell embryos in antiserum did not appear to
affect division of cells up to the 32-cell stage (Table 2, lines 1 and 2). Beyond
this stage, the length of exposure to antiserum influenced embryo cell numbers.
Embryos removed from antiserum prior to 84 h post-HCG did not differ
significantly in cell number from controls (lines 3-5, Table 2), whereas those
removed at later times had less cells than controls (lines 6-9, Table 2). The
mitotic index of embryos was also unaffected by exposure to antiserum for up
to 84 h post-HCG, but was reduced with further exposure to antiserum (Table 2).
3. Immunosurgery
Immunosurgery was used for two distinct purposes, (i) Embryos, which had
been grown continuously in antiserum, from the 2-cell stage for 48 h appeared
as decompacted bunches of cells. Control embryos had formed late morulae or
early blastocysts. Control embryos subjected to modified immunosurgery
(Handyside, 1978a) yielded well-defined clusters of inside cells. All the cells
of 43/44 antiserum-treated embryos were completely lysed. The one exception
(1/44) yielded only two live cells. Since both antibody and complement
incubations were carried out for only 5 min, and were washed prior to any
overt lysis, this result was taken as an indication of free access of both antibody
and complement to all cells of the antiserum-treated embryos.
(ii) Embryos, cultured in antiserum from the 2-cell stage for 48 h, were
rinsed, and cultured in medium 16 for a further 24 h, during which period
compaction occurred. The embryos were then subjected to modified immuno-
248
M. H. JOHNSON AND OTHERS
Fig.2(a)
FIGURE 2
(a) Electron micrograph of part of similar embryo to one shown in Fig. 1. Tight
junctional complexes were present between adjacent outer trophectodermal cells
(T) facing zona pellucida (Z), gap junctions between ICM cells (I) and overlying
trophectoderm and blastocoelic cavity (B). x 6589. (b) In higher magnification,
parallel arrays of longitudinally arranged microtubules (arrows) can be seen in
contact areas of adjacent trophectoderm cells (T) facing zona pellucida (Z), in the
process of formation of tight junctions, x 57456.
Role of compaction in blastocyst formation
249
Fig. 2(c). In serial section, microtubules are cut transversely (arrows). Zona pellucida
(Z) is seen in left corner, x 57456.
250
M. H. JOHNSON AND OTHERS
Fig. 3. Phase-contrast photomicrograph of mouse embryos recovered at the 2-cell
stage (45-46 h HCG) and cultured for 48 h in a 1 /40 dilution of an antiserum to
ec cells. Note absence of compacted appearance and of blastocoelic cavity but
presence of vesicles in individual cells, x 345.
surgery and the clusters of cells recovered were regarded as functionally inside
(although not necessarily ICM) cells. The brief exposure of fresh complement
ensured that any internal cells still coated with residual anti ec antibody were
not lysed secondarily to the outside cells. Whereas the ratio of inside: total cells
for control embryos was 0-23 (25 embryos), those for embryos incubated in
antiserum were 0-13 (35 true blastocysts), 0-006 (51 false blastocysts) and 0-01
(24 aggregate blastocysts).
4. Developmental properties of antiserum-treated embryos
Embryos, cultured in antiserum for 60 h, and then in medium 16 for 12 h,
were classified as being 'true', 'false' or 'aggregate' blastocysts and were
then washed and placed in RPMI +10 % foetal calf serum. Embryos were scored
at intervals for hatching, attachment, outgrowth and presence of an ICM-like
cell cluster on the outgrowth (Table 3). Since embryos classified as 'aggregate'
did not hatch, some of these embryos were freed from their zonas before culture.
These embryos attached and outgrew within 24 h and showed, in most cases, no
sign of an ICM-like cell cluster (Table 3).
Role of compaction in blastocyst formation
251
Fig. 4. Electron micrograph of part of similar embryo to one shown in Fig. 3. Note
absence of specialized junctions, vacuolar cytoplasm and blastocoelic cavity, x 3840.
EMB 54
252
M. H. JOHNSON AND OTHERS
Fig. 5. Phase-contrast photomicrograph of mouse embryo recovered at the 2-cell
stage (45-46 h HCG) and incubated in a 1/40 dilution of antiserum to ec cells for
60 h, followed by 12 h in medium 16 + BSA. The embryos have formed fluidaccumulating cell aggregates designated 'type 1' (see text), x 345.
Eight 'false' blastocysts transferred ectopically gave much smaller haemorrhagic nodules than control blastocysts. The nodules, on microscopic inspection,
revealed evidence of limited trophoblastic proliferation and transformation but
in only one case were egg cylinder remains detected. 'Aggregate' blastocysts did
not stimulate haemorrhagic nodules, regardless of whether or not the zonae
were removed prior to transfer.
Transfer of 12 'aggregate' blastocysts to pseudopregnant recipients resulted
in only one 'implantation site, with no decidual cell response and a ball of
trophoblastic giant cells occupying the uterine lumen. Of six 'true' blastocysts
transferred to pseudopregnant recipients, four implanted. Two implantation
FIGURE 6
(a) Electron micrograph of part of an embryo similar to that shown in Fig. 5. Note
that all cells contain vacuoles (arrows), there is some attempt at blastocoelic cavity
(BC) formation and junctional complex formation between long cytoplasmic strips
of cells. There are no obvious distinctive' inside cells', x 5426. (b) Membrane 'kisses'
(arrows) are seen between adjacent cells, x 29754.
Role of compaction in blastocyst formation
253
17-2
254
M. H. JOHNSON AND OTHERS
Fig. 7. Phase-contrast photomicrograph of an embryo treated as described in
legend to Fig. 5, but having formed a 'type-2' false blastocyst, in which the inside
cells mainly consist of a few fluid-accumulating cells, x 345.
chambers were devoid of live embryonic remains, one chamber contained a
dying embryo within a trophoblastic shell, and one contained a fully developed
embryo.
DISCUSSION
It has been proposed that compaction is a precondition for the generation of
the distinctive 'inside' and 'outside' environments thought to direct cell
differentiation in the morula (Ducibella, 1977), and various models for the
mechanism by which compaction could generate positional information have
been outlined (see Johnson, 1979 for detailed discussion). The experiments
reported here describe an antiserum which reversibly inhibits compaction
without obvious direct effects on cell division. The effect of the antiserum on
blastocyst differentiation depends upon the time for which it is permitted to
prolong decompaction.
Role of compaction in blastocyst formation
255
FIGURE 8
Electron micrograph of an embryo similar to those shown in Fig. 7. There is a
relative paucity of inside cells, x 4720. The inset shows the junctional complex
areas including the beginning of the formation of desmosome (arrows), x 28215.
256
M. H. JOHNSON AND OTHERS
2-cell
48
Mrs. post-HCG!
4-cell
Compaction
72
'
32-cell
early blastocyst
96
•
Expanded
blastocyst
ig
rr
' ago morphology
Normal True False
100
100
100
40
44
16
20
6
53
23
27
71
Fig. 9. Summary of protocol and results. Axis-hours post-HCG. Above axisdevelopmental state of embryos. Below axis - heavy bar = hours in anti-serum and
light bar = hours in control medium. Table at end indicates percentage of normal,
true, false and aggregate blastocysts for each treatment at 120 h post HCG.
1. Effect of decompaction up to the 32-cell stage
(i.e. removal from antiserum by 84 h post HCG)
The anti-ec antiserum successfully inhibits compaction of 8- to 32-cell mouse
embryos as judged by light and electron microscopic analysis and by the free
permeability of the intercellular spaces. The cells are devoid of the specialized
junctional complexes which normally develop over this period, but do remain in
physical contact within the cluster both by microvilli and close membrane
apposition. The antiserum appears to differ from that described by Kemler
et at. (1977) which inhibited compaction only when prepared as Fab fragments.
However, our preliminary studies showed that the dilution of antiserum was
critical for achieving a consistent effect on compaction and subsequent differentiation. The use of a critical 1/40 dilution almost certainly explains the lack of
requirement for a Fab fraction.
Our antiserum is undoubtedly directed against many specificities, and it is
not therefore possible to ascribe the blocked compaction to inactivation of any
specific membrane molecule. Whilst such a specific block may occur, the
antiserum could alternatively be affecting general cell surface properties, since it
is known that modification of general membrane structure and fluidity by
manipulation of membrane cholesterol also prevents compaction (Pratt, 1978).
Unlike other agents which block compaction, the antiserum does not appear
to affect the rate of cell division up to the 32-cell stage (94-96 h post-HCG) at
which time control embryos are forming blastocysts. Cytochalasin B (Ducibella,
1977), Cytochalasin D (Pratt, unpublished), colcemid (Pratt & Traver, unpublished), low Ca2+ (Reeve, unpublished) and reduced cholesterol (Pratt, 1978)
all suffer from the disadvantage that they restrict or block normal cytokinesis.
The fourth round of embryonic cell division, generating 16 cells, is the first
at which some cells become totally enclosed within the embryo (Barlow, Owen
& Graham, 1972; Handyside, 1978 b). Coincident with enclosure, comes cellular
118-120
94-96
Time of
examination
(h post HCG)
—
92 + 28(35)
—
—
—
—
—
—
—
—
—
—
29 ±7 (35)
(44)
—
—
—
—
—
—
—
2. Af(48)
3. C(72)
4. A(24) + C(48)
5. A(36) + C(36)
6. A(48) + C(24)
7. A(60) + C(12)
8. C(24) + A(48)
9. A(72)
63 ±16 (29)
(0-9)
78 + 19(5)
(2-8)
66
(2)
0-5)
—
—
—
—
—
—
'True'
blastocysts
* C = medium without antibody (h incubation).
t A =medium plus antibody (h incubation).
100±16(ll)
(2-5)
87+17 (7)
(2-6)
—
(3)
—
30 ±5 (20)
(3-2)
Blastocysts
—
Uncompacted
morulae
1. C*(48)
Incubation medium
Compacted
morulae
or early
blastocysts
62 + 15(13)
(10)
66+18 (10)
(17)
60+16(10)
(1-8)
50 ±8 (4)
(1)
(0)
—
—
—
—
—
'Aggregate'
blastocysts
66±13(24)
(09)
58 ±16 (9)
(10)
59 + 15(9)
(02)
57 + 16(6)
—
—
—
—
—
'False'
blastocysts
Cell no. in different embryonic forms ±S.E. (NO. of embryos examined)
(Mitotic index)
Table 2. Cell numbers in embryos grown from 2-cell stage (46-48 h post HCG) in medium ± anti ec (1/40)
©
ia
to
I
<**
©
3
S
1
s
o
258
M. H. JOHNSON AND OTHERS
Table 3. Development of embryos in vitro after culture from the 2-cell stage in
antiserum to ec teratoma (1/40) for 60 h followed by 12 h in medium 16, followed,
after selection and grouping by morphology, by 24 h in JRPM1
Control blastocysts
'Aggregate' blastocysts plus zona
pellucida
'Aggregate' blastocysts without
zona pellucida
'False blastocysts'
'True' blastocysts
No. of embryos showing
hatching, attachment and
outgrowth
No. of embryos showing
an ICM-like cluster on
the outgrowth
29/30
1/32
28/30
0/32
13/17
2/17
4/9
3/9
20/22
20/22
heterogeneity within the embryo (see Introduction), and the presumed segregation of ICM and trophectodermal cell lineages. In the absence of compaction
up to the 32-cell stage, cellular heterogeneity was not evident at the morphological level. However, when embryos were rinsed by 84 h post-HCG and restored,
to control media, compaction occurred within 10 h (i.e. by 94 h post-HCG) and
blastocysts formed, which could not be distinguished from control 120 h embryo.
It appears that the antibody exerts continuing effects on the embryo for
about 10 h after washing, as judged from recompaction times, so we can
conclude that a delay in compaction for up to 20 h beyond its normal time of
occurrence at 74 h post-HCG does not cause detectable modification of blastocyst
formation.
This apparent independence of blastocyst differentiation from a 20 h delay
in compaction is open to two interpretations. First, it could be argued that
compaction is irrelevant to blastocyst differentiation and that cells recognise
and respond to position in some manner that is independent of the compacted
state of the embryo. The distinctive effects of continuing incubation of embryos
in antiserum for periods beyond 84 h post-HCG argues against this interpretation, as does the apparent structural homogeneity of the cells in the 32-cell
decompact morula. Alternatively, it is possible that the process of compaction
is indeed critical to the generation of the positional cues which induce divergent
differentiation, but that over the 20 h period following normal compaction,
cells remain developmentally labile and able to oscillate between trophectodermal and ICM lineages as positional cues dictate. Evidence that this would be
possible has come from studies on the developmental lability of groups of inside
cells (Johnson, Handyside & Braude, 1977; Handy side, 1978a, b; Johnson, 1979)
and outside cells (Handyside, 19786) isolated from the late morula and early
blastocysts of the same strain as that used here. These studies showed that
inside cell lability started to fall off during blastocyst expansion at the same time
Role of compaction in blastocyst formation
259
that prolonged decompaction first starts to cause differentiative abnormalities
in the experiments described here.
Analysis of the data obtained from embryos incubated in antiserum beyond
84 h sheds further light on these alternative interpretations.
2. Effect of incubation in antiserum beyond 84-h post-HCG
When embryos are maintained in antiserum at progressively later times
beyond 84 h post-HCG, the proportion of normal blastocysts falls. Four types
of developmental abnormality are detected. (1) The morphology of the 'blastocyst' becomes increasingly abnormal. Whether or not the antiserum is washed
out during this period, the embryos appear to try to make blastocysts but the
proportion of 'aggregate' and 'false' blastocysts rises. Even in the 'true'
blastocysts the ICMs appear subjectively to be smaller, and direct measurements of inside: total cell numbers confirm this impression. (2) In all three forms
of blastocyst the total cell number is significantly reduced below that of the
controls which undergo one or two more rounds of division in contrast to only
part of a complete round of division in the antiserum-treated embryos.
(3) Functional tests of the 'blastocysts' suggest (i) that fluid-accumulating cells
consist largely or exclusively of trophectodermal cells, unable to hatch from their
zonas (Ansell & Snow, 1975) or proliferate, (ii) that false blastocysts consist of a
heterogeneous population some with ICMs and others without; in the false
blastocysts with ICMs, the ratio of inside cells to total is reduced, (iii) that true
blastocysts may also be less effective than controls at inducing successful
pregnancy or ectopic proliferation, either because of smaller total cell number or
a lower ratio of inside to total cells. (4) The mitotic index of all three forms of
blastocyst is reduced. Since the proportion of inside cells in all forms was lower,
the reduction in mitotic index could be explained by the absence or reduced
effectiveness of a proliferating ICM with lack of its consequent stimulation of
the overlying polar trpphectoderm (Gardner & Johnson, 1972).
If it is accepted that compaction is irrelevant to positional recognition in the
morula, the relative absence of ICM cells after prolonged exposure to antiserum would have to be explained by selective cell death. The cell death could
not be due simply to length of time in antiserum, since incubations started at the
8-cell stage and continued for only 48 h produced the same results as 72 h
incubations started earlier at the 2-cell stage. Moreover, brie would have to
assume that only relatively mature (and committed) ICM cells and not their
precursors "were sensitive. Although some cell death was observed in the electron
micrographs, it did not appear to be greater than that occurring in intact control
blastocysts (see also Copp, 1978).
The alternative explanation is that decompaction maintained beyond 94 h
post-HCG caused 'inside' cells which were previously developmentally labile, to
change irrevocably their developmental fate to a course of trophectodermal
differentiation. Any cells "which subsequently became trapped 'inside' a forming
260
M. H. JOHNSON AND OTHERS
blastocyst would nonetheless attempt to generate trophectodermal cells which
would explain some of the morphologies seen in the 'aggregate' and 'false'
blastocysts. The incompleteness of the diversion to trophectodermal development observed, particularly in those embryos washed free of antiserum at the
earlier periods, is consistent with the intrinsic heterogeneity of developmental
stage within a population of embryos of the same age post-HCG, as has been
observed by us using other parameters (Handyside & Johnson, 1978; Braude,
1979; Handyside, 1978 a, b).
In the absence of clear evidence for cell death in our embryos, we tend to
favour the second explanation. If it is accepted that cell fate is affected by prolonged decompaction, then this may indicate that the positional cues guiding
cells within the intact normal morula involve specialized cell junctions rather
than the general extent of simple cell contact (Johnson, 1979), since contact is
maintained in the decompacted embryos whereas specialized junction formation
is suppressed or delayed. This conclusion rests on the assumption that the
presence of antibody on the surface of the embryonic cells does not itself cause
gross distortion of normal cueing mechanisms. This is a considerable assumption, but these results do, at least, give an entry to the consideration of natural
cueing mechanisms and their relationship to cell differentiation and commitment in the late morula.
We wish to acknowledge the technical assistance of Gin Flach and Jo Close and the
valuable discussion with Dr Hester Pratt. The work was supported by grants from the M.R.C.
and the Ford Foundation to M. H. Johnson.
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{Received 23 May 1979, revised 13 August 1979)