/. Embryol. exp. Morph. Vol. 62, pp. 351-361, 1981
35]
Printed in Great Britain © Company of Biologists Limited 1981
Cytoplasmic polarity develops at compaction in
rat and mouse embryos
By W. J. D. REEVE 1
From the Department of Anatomy, University of Cambridge
SUMMARY
Cells of both rat and mouse morulae can be stained vitally to reveal an asymmetry in the
organization of their cytoplasm. In each cell of the rat 8-ceil embryo a column of organelles
develops between the nucleus and the embryo periphery as revealed by toluidine blue,
acridine orange and horseradish peroxidase (HRP). Although cells of the mouse morula lack
the blatant asymmetric distribution of organelles observed in rat cells, a long pulse (> 3 h) of
HRP to compact 8-cell mouse embryos revealed a distinct restricted localization of the
enzyme not evident at earlier pre-compaction stages. The cytoplasmic polarity generated in
these embryos can be demonstrated in cells of intact embryos, and also in cells disaggregated
from embryos before vital staining.
INTRODUCTION
The polarization hypothesis (Johnson, Pratt & Handyside, 1981) proposed
recently to explain the generation and maintenance of spatial differentiation in
the early preimplantation mouse embryo postulates the establishment in the
8-cell embryo of a radial asymmetry which is expressed in individual cells in the
form of an axial polarity. The generation of surface polarity in cells of the 8-cell
mouse embryo has been shown already (Handyside, 1980; Ziomek & Johnson,
1980; Reeve & Ziomek, 1981). This study demonstrates that cells of 8-cell rat
and mouse embryos also show a cytoplasmic polarity absent at earlier stages.
The existence of localized cytoplasmic material in the mammalian embryo
has long been proposed (Dalcq, 1957). Vital staining with toluidine blue
(Izquierdo, 1955) and acridine orange (Austin & Bishop, 1959), and studies by
transmission electron microscopy (TEM) (Izquierdo & Vial, 1962; Schlafke &
Enders, 1967) have shown a very clear reorganization of cytoplasmic components
at the 8-cell-stage of the rat embryo. Thus, whereas organelles of earlier stages
have a relatively uniform cytoplasmic distribution (Sotelo & Porter, 1959),
those of the 8-cell embryo are restricted largely to a column extending from the
nucleus of each cell to the embryo periphery (Izquierdo & Vial, 1962; Schlafke
& Enders, 1967; Dvorak, 1978). In contrast, the mouse embryo has been reported to display no such overt cytoplasmic polarization (Calarco & Brown,
1
Author's address: Department of Anatomy, Downing Street, Cambridge CB2 3DY, U.K.
12-2
352
W. J. D. REEVE
1969), although microtubules may orientate parallel to areas of cell contact, and
mitochondria occupy the cortical region (Ducibella, Ukena, Karnovsky &
Anderson, 1977). This report confirms that in the mouse 8-cell embryo, unlike
that of the rat, no blatant asymmetry is observed in the pattern of staining by
toluidine blue and acridine orange. In contrast, long pulses of horseradish
peroxidase (HRP) did result in a polarized distribution of ingested enzyme in the
cytoplasm of cells of compact morulae of both rat and mouse.
MATERIALS AND METHODS
Embryo collection
Female HC-CFLP mice (4-5 weeks; Hacking & Churchill) were superovulated with intraperitoneal injections of 5 i.u. of pregnant mare's serum
(PMS: Folligon, Intervet), followed after 44-48 h by 5 i.u. of human chorionic
gonadotrophin (hCG: Chorulon, Intervet). Females were paired with HC-CFLP
males, and vaginal plugs taken as an indication of mating. Mice were killed by
cervical dislocation and embryos were flushed from the oviducts, at times between
49 and 68 h post-hCG, with phosphate-buffered medium 1 supplemented with
0-4 % (w/v) bovine serum albumin (PB1 +0-4 % BSA) (Whittingham & Wales,
1969), and were cultured at 37 °C in medium 16 with 0-4 % (w/v) BSA (Whittingham, 1971) in 5 % CO2 in air.
Female Wistar rats (250 g body weight; Olac Limited) were paired with
Wistar males, and examined the next morning when spermatozoa in the vaginal
smear indicated day 1 of the pregnancy. Rats were killed by an ether overdose,
and embryos flushed from the oviducts with PB1+0-4% (w/v) BSA or pregassed culture medium, kept below pH 7-4. Four-cell embryos were obtained late
on day 3, and 8-cells during the afternoon of day 4. Embryos were cultured in
either a modification of the standard mouse medium of Biggers, Whitten &
Whittingham (1971) with 25 % (v/v) foetal calf serum (Mayer & Fritz, 1974) or
in T6' modified Tyrode's medium with 0-1 % (w/v) BSA (Wood & Whittingham,
1980).
Zonae pellucidae of both rat and mouse embryos were removed by a 15- to
30-sec incubation in prewarmed (37 °C) acid Tyrode's solution (pH 2-5) + 0-4 %
(w/v) polyvinylpyrrolidone (Nicolson, Yanagimachi & Yanagimachi, 1975).
Disaggregation
Disaggregation into single cells was accomplished by pipetting embryos with
a flame-polished micropipette after incubation in either trypsin/EDTA or
calcium-free medium, both pre-equilibrated for at least 30 min at 37 °C in 5 %
CO2 in air. After disaggregation, cells were restored immediately to the culture
medium. The disaggregating media were as follows.
(i) Trypsin/EDTA
Rat embryos were incubated in 0-5% (w/v) trypsin + 0-2% (w/v) EDTA
Cytoplasmic polarity at compaction in rat and mouse embryos
353
(Gibco) in calcium-free medium 16. After 5 min, when decompaction was
complete, the medium was drawn off and replaced with a large volume of
either PB1 +0-4 % (w/v) BSA, or pregassed culture medium.
(ii) Calcium-free medium
Mouse embryos were incubated for 10-30 min in calcium-free medium 16 +
0-6 % (w/v) BSA.
Toluidine blue
Cells or embryos were placed in a 1/50000 (w/v) solution of toluidine blue in
Tyrode's medium for 20 min before fixation in a solution formed from water and
saturated aqueous solutions of mercuric chloride, phosphotungstic acid and
ammonium molybdate in the volumetric ratio of 6:3:1:2 (Izquierdo, 1955).
After fixation for at least 1 h, embryos and cells were- washed in water, dehydrated through a graded alcohol series, cleared in toluene, and mounted in
Depex (Gurr Limited).
Acridine orange
Embryos and cells were incubated in a range of concentrations of acridine
orange (Gurr Limited) between 1/20000 (w/v) and 1/250 (w/v) in PB1 + 0-4 %
(w/v) BSA. The fluorescent staining pattern was observed directly, and 1/1000
(w/v) acridine orange gave optimum differential fluorescence. The viability of
compacted 8-cell embryos (67 h post-hCG) was examined by culture, either
continuously or for 2 h followed by restoration to control medium, in 1/1000
(w/v) acridine orange in medium 16 + 0-4% (w/v) BSA at 37 °C in 5 % CO2
in air.
Horseradish peroxidase (HRP)
Embryos and cells were incubated in 2 mg/ml HRP (Sigma Type II) in
medium 16 + 0-4% (w/v) BSA (mouse) or T6' modified Tyrode's medium +
0-1 % (w/v) BSA (rat) at 37 °C for 3-10 h in 5 % CO2 in air. Cells were rinsed
in PB1 +0-4 % (w/v) BSA, and fixed in 4 % (w/v) paraformaldehyde (Anderson
& Co. Ltd) in phosphate-buffered saline (PBS) at 4 °C for 1 h, before further
washing and storage in PB1 +0-4 % (w/v) BSA at 4 °C. Cells were stained for
HRP by the aminoethylcarbazole (AEC; Sigma) method (Pearse, 1968). Two
mg AEC were dissolved in 10 ml 50 mM acetate buffer pH 5-0 to which a drop of
3 % (v/v) hydrogen peroxide was added immediately before use. After staining
for 10-60 min, rat embryos were mounted in glycerine jelly (Cavanaugh, 1964),
while, for better resolution, mouse cells were examined in wells of a tissuetyping slide (Baird & Tatlock) in drops of PB1 +0-4 % (w/v) BSA under oil.
354
W. J. D. REEVE
Indirect immunofluorescence
HRP-treated embryos were incubated for 5 min in 25 fi\ drops of a rabbit
antiserum (RAMS) to mouse species antigens (Gardner & Johnson, 1975;
Handyside, 1980) diluted 1 in 15 in PB1+O4% (w/v) BSA + 0-02% (w/v)
sodium azide, followed by thorough washing in PB1+BSA + azide, and a
similar incubation in fluorescein-conjugated goat anti-rabbit IgG (FITC-GAR
igG; Miles Labs) diluted 1 in 15 in PB1+BSA + azide. The embryos were
washed again, and disaggregated into single cells which were fixed in 4 % (w/v)
paraformaldehyde in PBS at 4 °C for 1 h, before being stained for HRP.
Immunosurgery
Inner cell masses (ICMs) were isolated by immunosurgery (Solter & Knowles,
1975). Blastocysts were incubated for 5 min in RAMS (diluted 1:10 with PB1)
at 37 °C, washed extensively in PB1 +0-4 % (w/v) BSA, and incubated for 30
min in guinea-pig complement (Flow Labs.) (diluted 1:10 with PB1) at 37 °C.
After thorough washing, the inner cell masses were separated from the lysed
trophectodermal cells by drawing the embryos through a finely pulled Pasteur
pipette.
Cell counting
Embryos and inner cell masses were prepared and fixed according to
Tarkowski (1966). Nuclei were stained with a millipored 1 % (w/v) suspension
of Giemsa (Raymond A. Lamb). Cell numbers were not recorded for the very
small minority of spreads which showed either excessive clustering or dispersion
of nuclei.
Light microscopy
A Zeiss Universal microscope, which was fitted with incident source HBO
50, III RS condenser and Zeiss filter set 487709, was used to examine cells for
acridine orange staining and HRP-stained cells for FITC-labelling. Kodak
Tri-X 35 mm film was used for both bright-field and fluorescence photography.
Cells mounted in Depex or glycerine jelly were examined with a Zeiss Ultraphot
II microscope, and photographed on Pan F film.
Transmission electron microscopy
Embryos were fixed for 1 h at room temperature in 2-5 % (v/v) glutaraldehyde
in 0-1 M sodium cacodylate buffer at pH 7-4. Embryos were washed with the
buffer, and postfixed in 1 % (w/v) osmium tetroxide in 0-1 M sodium cacodylate
buffer. After dehydration through a graded alcohol series, embryos were infiltrated and embedded in Spurr resin (Spurr, 1969). Sections, 50 nm thick, were
cut with a glass knife, and stained with uranyl acetate (Gibbons & Grimstone,
1960) followed by lead citrate (Reynolds, 1963). Sections were viewed in a
Siemens Elmiskop I microscope.
Cytoplasmic polarity at compaction in rat and mouse embryos
355
RESULTS
1. Toluidine blue
Vital staining of rat embryos with toluidine blue revealed uniform cytoplasmic
staining in 4-cell blastomeres (Fig. 1) and a polarized pattern in 8-cell blastomeres (Fig. 2). The metachromasia with toluidine blue is considered by Izquierdo
& Vial (1962) to be associated with vesicles which are rich in mucopolysaccharides and acid hydrolases (Stastna, 1974). At the 8-cell stage there is a
segregation of organelles into a column extending from the nucleus of each cell
to the embryo periphery (Fig. 3). Five different staining patterns were identified
in single cells disaggregated from embryos and then stained (Fig. 4), When
classified according to staining pattern, cells from 8-cell embryos showed a high
incidence of cytoplasmic polarity compared with cells from 4-cell embryos
which lacked cytoplasmic columns (Table 1). However, when all cells of individual embryos were examined, non-polarized cells were detected in many
embryos in which the majority of cells were polarized (W. J. D. Reeve, unpublished observations). Culture of both intact embryos and single cells in
vitro before staining resulted in a slight decrease in the incidence of polarity
(Table 1). Tn contrast to the rat embryo, cells from 8-cell mouse embryos stained
with toluidine blue offered only a hint of polarity which was lost after fixation.
2. A cridine orange
Acridine orange stains the DNA of the nucleus green, and lysosomes and
ribonucleic acids are considered to be associated with the orange cytoplasmic
staining (Austin & Bishop, 1959; Allison & Young, 1969). Staining of intact
8-cell rat embryos and their dissociated cells revealed asymmetries of cytoplasm
similar to those observed with toluidine blue. After being stained with acridine
orange, dissociated rat cells were sorted on the basis of cytoplasmic polarization,
and then further stained with toluidine blue. The two stains were localized in
similar patterns. Thus, of 16 cells which stained with acridine orange to reveal
a column stretching from the nucleus to the cell surface, all showed columns
after staining with toluidine blue; seven cells had nuclear caps or rings
when stained with both acridine orange and toluidine blue; and of four cells
shown by acridine orange to have a scattered distribution of organelles, staining
with toluidine blue showed two to have nuclear caps, one to have a uniform
staining pattern and one to be unscorable. In contrast, only a minority of
dissociated cells of both pre-compact and compact mouse embryos revealed an
asymmetry of cytoplasmic staining with acridine orange over a range of concentrations. Just 16 % of 53 cells from pre-compact 8-cell embryos and 9 % of
64 cells from compact 8-cell embryos revealed a columnar cytoplasmic staining
pattern. There was a similar low incidence of cytoplasmic stain concentrated in
nuclear caps, nuclear rings and dispersed cytoplasmic aggregates. In contrast,
356
W. J. D. REEVE
2
1
it-
50
50
Fig. 1. The 4-cell rat embryo shows uniform cytoplasmic staining after immersion in
toluidine blue.
Fig. 2. An 8-cell rat embryo stained with toluidine blue reveals a column extending
from the nucleus of each cell to the embryo periphery.
Fig. 3. The region of intense staining corresponds to columns of organelles seen in
TEM (x 17000), N, nucleus; Z, zona pellucida; C, column of organelles.
Cytoplasmic polarity at compaction in rat and mouse embryos 357
4a
4b
Ac
f
4c/
4e
,
3 0 Mm
Fig. 4. Rat 8-cell embryos were disaggregated to single cells and stained with toluidine blue. Five categories of staining pattern were recognized, (a) Tight column.
Metachromasia extends in band (width less than nuclear diameter) from the nucleus
to the periphery of the cell, (b) Loose column. The column is wider than the nucleus.
(c) Nuclear cap. The metachromasia extends less than half-way from the nucleus to
the cell periphery, (d) Nuclear ring. Metachromasia surrounds the nucleus, (e)
Uniform stain. Stain occurs throughout the cytoplasm.
53 % and 66 % of the cells from pre-compact and compact 8-cell embryos,
respectively, showed a uniform cytoplasmic pattern of staining.
3. Horseradish peroxidase
Incubation with HRP revealed a polarized HRP accumulation in compact rat
embryos (Fig. 5) and a similar pattern was also found in cells from compact
mouse morulae (Figs 8, 9). All cells in any one embryo showed very similar
patterns of stain distribution. Thus the staining patterns of intact embryos were
classified as either uniform, localized or aggregated. Embryos showing a
localized pattern of staining appeared to have one restricted mass of stain near
the nucleus of each cell, while embryos classed as aggregated appeared to have a
more diffuse distribution of HRP-containing vesicles. Mouse embryos, which
were either 4-cells (Fig. 6) or pre-compact 8-cells (Fig. 7) at the termination of
pulses of HRP of up to 10 h duration, did not show a restricted localization of
HRP-containing vesicles. The incidence of localized HRP-containing vesicles
14.1 ±2.6
(7)
8.0 ±1.3
(81)
8.4±1.6
(33)
9.6 ±3.1
(33)
4.0 ± 0
(10)
4.0 ± 0
(9)
7
231
10
11
143
80
19
0
35
431
0
37
Tight
column
44
23
37
47
0
0
Loose
column
18
22
18
12
12
0
Nuclear
cap
Cytoplasmic staining*
21
16
15
8
14
38
7
32
19
14
74
62
19
27
33
21
3
8
93
68
81
86
26
38
Nuclear
% Unscored
ring
Uniform or lost cells % Polarizedf
* Use of these staging criteria may cause an underestimate of the incidence of polarization since a cytoplasmic column perpendicular to the
focal plane cannot always be identified.
t % Polarized = % of scored cells which lacked a uniform pattern of cytoplasmic staining.
Immediate disaggregation
and staining
Intact embryos cultured
in vitro 10 h before
disaggregation and
staining
Immediate disaggregation
and staining
Single cells cultured
in vitro 7 h before staining
Intact embryos cultured
in vitro 7 h before
disaggregation and staining
Intact embryos retained
in vivo for 7 h before
disaggregation and staining
Treatment
Mean number
of cells per
embryo ± S.D.
Number of
(number of embryos) scored cells
Table 1. Cytoplasmic staining patterns with toluidine blue of cells dissociated from rat embryos before staining
w
oo
Cytoplasmic polarity at compaction in rat and mouse embryos
359
increased in the more overtly compact embryos (Table 2). Whereas 64 % of precompact 8-cell embryos had cells which showed a uniform distribution of HRPcontaining vesicles, only 14 % of compact embryos had cells which did so.
Disaggregation of pre-labelled individual compact embryos showed that
restricted localization can occur in all 8 cells of an embryo, although one or two
cells in any one embryo may not appear polarized. Double staining with HRP
and fluorescent antibody demonstrated that the localization of HRP lies under
the fluorescent pole described previously (Fig. 9) (Handyside, 1980; Ziomek &
Johnson, 1980; Reeve & Ziomek, 1981). The incidences of the different patterns
of localization of HRP-containing vesicles (Fig. 10) were similar for cells
labelled in situ before disaggregation, and for cells from embryos completely
disaggregated before incubation with HRP (Table 3).
4. Viability of embryos
Mouse embryos cultured in toluidine blue lysed within 1 h, and acridine
orange also proved harmful to development. Compacted 8-cell embryos (67 h
post-hCG), whether exposed to acridine orange for 2 h or continuously, retained
a compact appearance at 100 h post-hCG when control embryos were expanded
blastocysts. At 125 h post-hCG, all embryos pulsed earlier for 2 h with acridine
orange had formed abnormal blastocysts with extruded cells, while morulae
still in acridine orange had lysed. Prolonged culture in HRP did not affect
development so adversely (Table 4).
DISCUSSION
Cells of rat and mouse 8-cell embryos show evidence of a cytoplasmic polarity
absent at earlier stages of development. The results for the rat embryo confirm
and extend previous observations, but hitherto no equivalent polarity in cells
of the mouse embryo has been described.
There is abundant cytological evidence from light and electron microscopic
studies on rat embryos to support the results obtained by staining with toluidine
blue and acridine orange. In the rat 4-cell embryo, organelles lose the scattered
distribution of earlier stages (Sotelo & Porter, 1959), and tend to localize at the
periphery and around the nucleus (Mazanec & Dvorak, 1963; Schlafke &
Enders, 1967; Stastna, 1974). At the 8-cell stage, each cell shows a definite
cytoplasmic segregation involving the organization of almost all organelles into
a column extending from the nucleus to the embryo periphery. The column
contains most of the mitochondria, the small regions of the Golgi apparatus, the
endoplasmic reticulum (mostly agranular) and a heterogeneous assortment of
vesicles (Izquierdo & Vial, 1962; Schlafke & Enders, 1967; Stastna, 1974). These
cytoplasmic columns are revealed by vital staining with both toluidine blue
(Fig. 2) (Tzquierdo, 1955) and acridine orange (Austin & Bishop, 1959). Outside
the column, extensive areas of more dense homogeneous cytoplasm contain a
360
W. J. D. REEVE
\
'v.
50 um
50
50
50
f
, 30 um
f
0
30 Mm .
Cytoplasmic polarity at compaction in rat and mouse embryos
361
Table 2. Incidence of HRP staining patterns in intact %-cell mouse embryos
Stage of
embryos
Total
number of
embryos
Pre-compact
91
Peri-compact
Compact
69
65
Uniform
Staining pattern*
Cytoplasmic
aggregates
Restricted
localization
(%)
(%)
(%)
58
21
12
(64)
(23)
(13)
29
25
15
(42)
(36)
(22)
9
20
36
(14)
(31)
(55)
* All cells in any one embryo showed very similar patterns of stain distribution.
few mitochondria and vesicles, and large amounts of proteinaceous lamellae
considered to be storage material used in cleavage (Dvorak et al. 1975; Dvorak,
Travnik & Stankova, 1977). The columns are stable, and persist even in isolated
cells cultured for several hours (Table 1). There are, however, problems in the
use of the rat embryo for a dynamic study of polarization. First, the supply of
embryos is limited as superovulation in the rat is not an established technique.
Second, published data on the culture of preimplantation rat embryos is
scarce, and successful culture difficult. Although culture of 8-cell embryos to
blastocysts has been reported as 80 % or more successful (Folstad, Bennet &
Dorfman, 1969; Mayer & Fritz, 1974), the media used by these authors and also
the T6' medium of Wood & Whittingham (1980) never gave a success rate above
60 % (W. J. D. Reeve, unpublished results). Lastly, development in vitro of the
rat embryo over the 4-cell stage is particularly difficult (Suzuki & Iizuka, 1969;
Mayer & Fritz, 1974), thus preventing culture over the period in which cytoplasmic polarity is generated.
The compact mouse embryo lacks the blatant cytoplasmic segregation
described for the rat 8-cell embryo when examined by TEM (Calarco & Brown,
FIGURES
5-9
Fig. 5. A rat morula stained with HRP to show cytoplasmic polarity. Individual cells
which show a polarized distribution of HRP are arrowed. (The other cells also show
a restricted localization of the enzyme, but are not in focus.)
Fig. 6. A mouse 4-cell embryo shows a uniform cytocortical distribution of HRP.
Fig. 7. A pre-compact 8-cell mouse embryo shows widespread distribution of HRP.
Fig. 8. A compact 8-cell mouse embryo shows the restricted cytoplasmic localization
of HRP peripheral to the nucleus of each cell. Two cells are arrowed.
Fig. 9. Cells of an 8-cell mouse embryo stained with (a) HRP and (b) fluorescent
ligand before embryo disaggregation. Each pole of fluorescent ligand-binding overlies the restricted cytoplasmic localization.
362
W. J. D. REEVE
Table 3. Incidence of staining patterns in cells of compact S-cell mouse embryos
stained with HRP (68-72 h post-hCG) before or after disaggregation
Treatment
Disaggregation
before staining
Disaggregation
after staining
Total
Tight
cell
localnumber ization
Loose
localization
Nuclear
ring
Aggregates
Uniform Unscorable
81
21
26
8
17
6
3
80
25
23
7
18
3
4
1969), although showing a surface polarization of microvilli (Ducibella et al.
1977; Reeve & Ziomek, 1981), numerous microtubules orientated parallel to the
apposed membranes of blastomeres, and mitochondria localized to the cortex
(Ducibella et al. 1977). Neither toluidine blue nor acridine orange staining
patterns provided conclusive evidence of cytoplasmic polarity in blastomeres of
compact 8-cell mouse embryos.
In contrast, when HRP was used as a vital stain, cells of both rat and mouse
8-cell embryos showed a pronounced cytoplasmic polarity. HRP differs from
acridine orange and toluidine blue in its active uptake by cells, and its dependence on cellular mechanisms for transport, ultimate localization and metabolism. It has proved a useful tracer in studies of endocytosis (reviewed Silverstein, Steinman & Cohn, 1977) as it is non-toxic and its enzymic activity can be
demonstrated histochemically (Graham & Karnovsky, 1966). HRP uptake is by
fluid pinocytosis, and washing before fixation ensures that adsorbed enzyme is
removed from the cell surface (Steinman & Cohn, 1972; Steinman, Silver &
Cohn, 1974). In published data on preimplantation rabbit (Hastings & Enders,
1974) and rat (Schlafke & Enders, 1972) embryos, HRP pulses never exceeded
60 min, and provided mostly information on uptake patterns at the cell surface.
Few vesicles were observed before the 8-cell stage, and endocytosis increased
by the blastocyst stage, at which vesicles were restricted mainly to the supranuclear region.
Four-cell mouse embryos showed a uniform distribution of HRP reaction
product in the cytocortex, but poor cytoplasmic staining, after prolonged pulses
of HRP (Fig. 6). However, after pulses as short as 3 h, some 8-cell embryos were
shown to have localization of reaction product between the nucleus of each cell
and the embryo periphery (Figs. 8, 9). The increase in the incidence of dispersed
aggregates and of restricted localization of HRP stain in cells was associated with
compaction (Table 2). The restricted localization of HRP-containing vesicles
shown by a minority of pre-compact 8-cell embryos is consistent with the generation of surface polarity before overt cell flattening (Ziomek & Johnson, 1980;
Reeve & Ziomek, 1981). Interestingly, in the rat embryo, compaction as assessed
26
17
31
29
Culture conditions
Control
+ HRP
*ICM/blasto
Number of
blastocysts
Initial number
2-cell embryos
~ "*
mean cell inumber of ICMs
mean cell number of blastocysts.
19-5 ±7-2
(11)
21-3 ±6-8
(8)
84-5 ± 32-2
05)
68-0 ±181
(9)
J
Inner cell mass (ICM)
mean cell no. ± S.D.
(no. ICMs)
Blastocyst
mean cell no. ± S.D.
(no. blastocysts)
0 31
0-23
ICM/blastocyst*
Table 4. Toxicity of HRP for cultured 2-cell mouse embryos (49 h post-hCG)
V
V
Outgrowth
s
Hi
r
•§
364
W. J. D. REEVE
10a
106
•
10c
10c/
*
»
10e
30 urn
Fig. 10. Dissociated cells of mouse 8-cell embryos showed several patterns of localization of HRP vesicles, (a) Tight localization, (b) Loose localization, (c) Nuclear
ring, (d) Aggregates, (e) Uniform.
by cell flattening occurs at the 4-cell stage, although the cytoplasmic organelles
become organized into columns only at the 8-cell stage. The polarity of HRP
localization in the intact 8-cell mouse embryo does not depend on differences in
the area of exposed cell surface, since it is also shown by dissociated cells incubated in HRP (Table 3).
There is no obvious ultrastructural basis for the restricted localization of
HRP stain. The HRP reaction product is thought to coincide with the Golgi
apparatus (Steinman et al. 1974; Piasek & Thyberg, 1979), but information on
the Golgi apparatus of the rodent embryo is confined almost entirely to the rat.
The quantity of Golgi changes little during cleavage stages (Dvorak et al. 1977).
Cytoplasmic polarity at compaction in rat and mouse embryos
365
Although early cleavage stages were shown to have Golgi zones near both the
cell membrane and nucleus, in the 8-cell embryo the membranous components
are aggregated and small Golgi zones are present throughout the organelle-rich
regions of cytoplasm (Schlafke & Enders, 1967). However, Stastna (1978)
reported no obvious relationship between the Golgi apparatus and the nucleus or
blastomere surface in cleavage-stage embryos. The Golgi complex of the preimplantation mouse embryo is not prominent. The occasional small regions of
stacked cisternae in early cleavage stages develop to larger regions of stacked
cisternae in the morula (Calarco & Brown, 1969). The Golgi complex of the
blastocyst of both the rat (Schlafke & Enders, 1963, 1967; Stastna, 1972, 1974,
1978) and the mouse (Calarco & Brown, 1969; Nadijcka & Hillman, 1974)
embryo is located in a juxta-nuclear position.
The demonstration that mouse 8-cell blastomeres are cytoplasmically polarized
on an identical axis to the surface polarity already described (Handyside, 1980;
Ziomek & Johnson, 1980; Reeve & Ziomek, 1981) suggests a reorganization of
cell structures and function at this developmental stage. Such a reorganization is
consistent with the first postulate of the polarization hypothesis (Johnson, Pratt
& Handyside, 1981) which suggests that the operation of axial polarity in 8-cell
blastomeres involves the allocation of ICM- and trophectoderm-like properties
to basal and apical portions, respectively, of the cell. Distinct cell lineages could
then be generated by subsequent divisions of the polarized cells. An asymmetric
cellular distribution at division has already been demonstrated for features of
surface polarization (Johnson & Ziomek, 1980), and the stability and lack of
toxicity of HRP may permit a similar analysis of the conservation of cytoplasmic
polarity.
I am grateful to John Bashford and Ian Edgar for technical help, and to Drs M. H. Johnson,
C. A. Ziomek and A. H. Handyside for valuable discussion. This work was supported by
grants from the Medical Research Council and the Ford Foundation to Dr Johnson, and by
an MRC research studentship.
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600-628. Amsterdam: North-Holland.
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{Received 1 August 1980, revised 20 October 1980)