J. Embryo!, exp. Morph. Vol. 66,pp. 191-207,1981
Printed in Great Britain © Company of Biologists Limited 1981
\()\
The distribution of ingested horseradish peroxidase
in the 16-cell mouse embryo
By W. J. D. REEVE 1
From the Department of Anatomy, University of Cambridge
SUMMARY
Cells of the 16-cell mouse embryo endocytose horseradish peroxidase (HRP) which
becomes localized in most cases to a juxtanuclear position. Cells that have ingested HRP
in intact embryos, and cells dissociated from embryos prior to culture in HRP, showed
similar patterns of cytoplasmic distribution of the ingested enzyme. Cells in the embryo in
situ were incubated in HRP, and then labelled with fluorescent antibody either before (to
label the outside surface of the embryo) or after (to reveal populations of outer polar and
inner apolar cells) their disaggregation into single cells. The population of polar outside
cells from the morula includes more cells with a highly restricted localization of HRPcontaining vesicles than does the population of inside cells, and this restricted localization
underlies the exposed surface or pole of the cell. A 2/16 couplet formed by division in vitro
of a 1/8 cell is comparable to the pairs of cells dissociated from 16-cell embryos; most
couplets from either source consisted of a larger cell that showed polarized surface binding of
fluorescent ligand (fluorescent pole) and a smaller cell with a uniform distribution of bound
ligand. The incidence of restricted patterns of HRP staining was highest among populations
of both larger and polar cells. When 1 /8 cells labelled with HRP are observed during division
to 2/16, the previously clustered vesicles of ingested HRP become more dispersed throughout
the cytoplasm and, although the two cells of some couplets can stain differently very soon
after their formation, the patterns of distribution of HRP take about 1 h after division to
stabilize. These observations are consistent with cells of the 16-cell embryo inheriting different
features of cytoplasmic organization.
INTRODUCTION
Cellular position within the compacted mouse morula has long been thought
to be critical for the differentiation of the inner cell mass (ICM) and trophectodermal lineages of the blastocyst (Tarkowski & Wroblewska, 1967; Herbert &
Graham, 1974; Ducibella & Anderson, 1975; Kelly, Mulnard & Graham,
1978). The mechanism by which position is translated to fate has been unclear.
Recently, it has been proposed that the fate of cells within the 16-cell morula
does not depend solely on their position within the embryo (Johnson, Pratt &
Handyside, 1981). Each cell of the 8-cell embryo becomes polarized along the
radial axis of the embryo, and a differential inheritance at division to the 16-cell
stage generates two cell types (Johnson & Ziomek, 1981): one present on the
1
Author's address: Department of Anatomy, Downing Street, Cambridge CB2 3DY,
U.K.
7-2
192
W. J. D. REEVE
inside, the other on the outside of the morula (Handyside, 1981; Reeve &
Ziomek, 1981). The polarity of individual cells of the 8-cell embryo has been
shown by asymmetric distributions of surface-bound fluorescent ligand
(Handyside, 1980; Ziomek & Johnson, 1980) and microvilli (Reeve & Ziomek,
1981), the high incidence of mitochondria and microtubules orientated parallel
to the cell surface near areas of cell apposition (Ducibella, Ukena, Karnovsky &
Anderson, 1977), alkaline phosphatase activity confined to apposed cell surfaces
(Mulnard & Huygens, 1978), the basal location of the nucleus of each cell
(Reeve, unpublished) and the restricted cytoplasmic localization of endocytosed
vesicles containing horseradish peroxidase (HRP) (Reeve, 1981).
Sixteen-cell embryos can be labelled with a variety of fluorescent ligands
either before or after their disaggregation into single cells. Immunofluorescent
labelling of embryos before disaggregation marks those cell surfaces which are
part of the external surface of the embryo, whereas, after disaggregation, some
isolated cells show a polarity of ligand binding associated with a heterogeneity
of microvillous distribution. Thus, in the 16-cell embryo, the inside cells have
an even distribution of sparse microvilli and bind fluorescent ligand uniformly,
while each outside cell has a region of dense microvilli (microvillous pole) facing
outwards and associated with a high level of ligand-binding sites (Handyside,
1981; Reeve & Ziomek, 1981; Johnson & Ziomek, 1981). Most polarized cells
of the 8-cell embryo divide, both in the embryo in situ (Handyside, 1981;
Johnson & Ziomek, 1981) and in isolation (Johnson & Ziomek, 1981), to give
one polar cell and one apolar cell.
This paper extends the description of the patterns of cytoplasmic localization
of endocytosed HRP in the 8-cell embryo (Reeve, 1981) to the 16-cell stage at
which it is demonstrated that inside and outside cell populations show different
patterns of distribution of the ingested HRP. The tightly localized pattern of
HRP distribution is more frequent in polar outside cells than in apolar inside
cells, and occurs on the same axis as the surface polarity, a correlation described
previously for the 8-cell embryo (Reeve, 1981). The patterns of HRP-vesicle
localization are affected by both cell size and the presence of a microvillous pole,
and the difference in staining patterns between polar and apolar cells is shown
by the cells in the 16-cell embryo in situ, and by 2/16 couplets generated by
division in vitro of 1/8 cells. Thus, cells from the 16-cell stage appear intrinsically
different from the time of their formation at division, and these differences
can occur in the absence of the inside and outside environmental cues previously considered critical to differentiation in the preimplantation mouse
embryo (Ducibella & Anderson, 1975).
Cytoplasmic organization in the mouse morula
193
MATERIALS AND METHODS
Embryo collection
Female HC-CFLP mice (4-5 weeks; Hacking & Churchill) were superovulated
with intraperitoneal injections of 5 i.u. 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
the presence of vaginal plugs taken as an indication of mating. Embryos were
flushed from the oviducts at 66-70 h post-hCG with phosphate-buffered
medium 1 supplemented with 0-4% (w/v) bovine serum albumin (PB1 + BSA)
(Whittingham & Wales, 1969), and were cultured at 37 °C in medium 16 with
0-4% (w/v) BSA (M16 + BSA) (Whittingham, 1971) under paraffin oil in 5 %
CO2 in air.
Zonae pellucidae were removed by a 15-30 s incubation in prewarmed
(37 °C) acid Tyrode's solution (pH 2-5) + 0-4% (w/v) polyvinylpyrrolidone
(Nicolson, Yanagimachi & Yanagimachi, 1975).
Terminology
Throughout this paper, the individual cells of 8-cell embryos are called
1/8 cells, and those of 16-cell embryos, 1/16 cells. Thus, an isolated 1/8 cell
divides in vitro to form a 2/16 couplet.
Disaggregation and decompaction
Disaggregation into single cells was accomplished by pipetting embryos with
a flame-polished micropipette after incubation for 10-30 min in calcium-free
medium 16 + 0-6% (w/v) BSA, 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.
2/16 couplets that had compacted were decompacted by incubation for
5 min in calcium-free medium 16 + 0-6% (w/v) BSA.
Horseradish peroxidase (HRP)
Embryos and isolated cells were incubated in 2 mg/ml HRP (Sigma Type II)
in M16 + BSA for 3-10 h in 5 % CO2 in air. Embryos were then disaggregated
for examination of the patterns of HRP staining of cells cultured in HRP in
the embryo in situ. Intact embryos and isolated cells were rinsed in PB1 + BSA,
and fixed in 4 % (w/v) paraformaldehyde (Anderson & Co. Ltd) in phosphatebuffered saline (PBS) at 4 °C for 1 h, before further washing and storage in
PB1 +BSA at 4 °C. Cells were stained histochemically for HRP by the aminoethylcarbazole (AEC; Sigma) method (Pearse, 1968; Reeve, 1981), and then
mounted in wells of a tissue-typing slide (Baird & Tatlock) in drops of PB1 +
BSA under oil.
194
W. J. D. REEVE
Couplets
A population of dissociated 1/8 blastomeres was cultured for at least 3 h
in 2 mg/ml HRP in M16 + BSA, and 2/16 couplets were then harvested at
20 min or 1 h intervals. The couplets remained in the HRP-containing medium
for various defined times until later immunofluorescent labelling and fixation.
The division in vitro of a 1/8 cell to a 2/16 couplet has been shown to be comparable to the equivalent division in the intact embryo, as assessed by comparison of surface features (Johnson & Ziomek, 1981).
Indirect immunofluorescence
HRP-treated embryos and cells were incubated for 5 min in 25 /i\ drops of
rabbit antiserum (RAMS) to mouse species antigens (Gardner & Johnson,
1975) diluted 1 in 15 in PB1 +BSA + 0-02% (w/v) sodium azide, followed by
thorough washing in PB1 + BSA + azide, and a similar incubation in fluoresceinconjugated goat anti-rabbit IgG (FITC-GAR IgG; Miles Labs.) diluted 1 in 15
in PB1 + BSA + azide. Embryos and cells were washed again, and embryos
disaggregated into single cells. The couplets and single cells were fixed in
4 % (w/v) paraformaldehyde in PBS at 4 °C for 1 h, before being stained for
HRP activity.
Fluorescence microscopy
A Zeiss Universal microscope, fitted with incident source HBO 50 and Zeiss
filter set 487709, plus additional excitation filter LP 425, was used to examine
cells for patterns of HRP staining and F1TC labelling. Bright-field and fluorescent micrographs were taken with Kodak Tri-X 35 mm film.
RESULTS
(1) The distribution of ingested HRP in cells of intact embryos
Intact 16-cell embryos were cultured in HRP, and then labelled by indirect
immunofluorescence either before or after disaggregation to single cells or
couplets of cells. Fluorescent labelling before disaggregation permitted comparison of the HRP staining patterns of inside (unlabelled by fluorescence) and
outside (labelled by fluorescence) cells, whilst labelling of dissociated cells
allowed examination of the HRP localization patterns of apolar (inside) and
polar (outside) cells. A mean of 6 0 cells/embryo occupied an inside position
as revealed by lack of fluorescence on cells from embryos labelled with fluorescent ligand before their disaggregation to single cells; whereas, for those
embryos disaggregated completely before fluorescent labelling, 6-7 cells/embryo
occupied an inside position as shown by a non-polarized binding of ligand
(Table 1; lines 1 and 2). These figures are similar to those reported previously
(Johnson & Ziomek, 1981; Handyside, 1981).
Cytoplasmic organization in the mouse morula
195
.*",'*«! 1 *
Fig. 1. The scale bar = 30 /*m. The cells of the 16-cell embryo showed several
staining patterns, (a) Tight localization, (b) Loose localization, (c) Horseshoe.
(d)2-poles. Two aggregates of HRP occur on opposite sides of the nucleus, (e) Nuclear
cap. (/) Nuclear ring, (g) Aggregates or uniform.
Several patterns of H R P distribution in individual cells were identified
(Fig. 1). The patterns included (Fig. 1 a) a 'tight localization' with HRP-vesicles
concentrated in one large well-defined mass; (Fig. \b) 'loose localization' in
which diffuse aggregates of staining occupied a small cytoplasmic region;
(Fig. 1 c) 'horseshoe' in which approximately half of the nucleus was surrounded
by staining; (Fig. 1 d) '2-poles' shown by two well-defined small masses of HRP
on opposite sides of the nucleus; (Fig. 1 e) 'nuclear cap' in which a small area of
intense staining was juxtaposed to the nucleus, and (Fig. l / ) a 'nuclear ring'
revealed by staining concentrated around the nucleus. 'Aggregates' or uniform
patterns (Fig. 1 g) appeared as diffused distributions of vesicles throughout the
cytoplasm. The tight and loose localization patterns represent large restricted
areas of staining, and were more common in outside polar cells than in inside
apolar cells (Table 1; lines 1 and 2). Cells showing these two patterns are
hereafter defined as showing 'restricted' localization. The other patterns of
stain distribution were grouped as 'non-restricted'. In outside cells, the restricted cytoplasmic localization of HRP occurred on the same a*xis as, and underlay, the fluorescent surface labelling on the outward-facing surface of the cell
(Fig. 2).
Total
419
430
292
27
29
—
262
(63)
249
(58)
172
(59)
P*
157
(37)
181
(42)
120
(41)
AP*
—
6-7
60
Calculation of
mean no. nonpolar cells
per embryo
164
(63)
154
(62)
135
(78)
P
70
(45)
74
(41)
46
(38)
AP
98
(37)
95
(38)
37
(22)
P
87
(55)
107
(59)
74
(62)
AP
Restricted Non-restricted
* P = polar; AP = apolar patterns of surface-labelling with fluorescentligand.
t Derived by division in vitro of 1/8 cells.
Antibody label before disaggregation
of embryos
Antibody label after disaggregation
of embryos
Antibody-labelled coupletsf
Treatment
No. of
embryos
No. of cells
(% of total)
Cytoplasmic staining
patterns
(% of totals)
Table 1. Cytoplasmic staining patterns in cells from intact 16-cell embryo (70-74 h post-hCG) and
2/16 couplets stained with HRP
REEVE
Cytoplasmic organization in the mouse morula
197
Fig. 2. The scale bar = 30urn. All cells of a 16-cell embryo incubated in (a) HRP
and (b) labelled withfluorescentligand before embryo disaggregation. The isolated
cells were then fixed and stained histochemically. The 11 outside cells tend to show
greater localization of HRP than the 5 inside cells, and this localization underlies
the pole of surface fluorescent labelling.
(2) The restricted localization of HRP does not depend on the restricted exposure
of the cell surface
The incidences of the different patterns of staining did not differ significantly
whether cells were incubated in HRP in embryos in situ before disaggregation
of the embryos to single cells, or dissociated from embryos before their culture
in HRP (Table 2). Thus, the difference in HRP localization patterns is not
obviously related to differential accessibility of the cell surfaces to HRP, and
this conclusion is supported by observations on 2/16 cells generated by division
of isolated 1/8 cells in vitro.
(3) The cytoplasmic staining patterns of 1/16 cells from intact embryos cultured
in HRP are similar to those of 1/16 cells generated by division of isolated 1/8
cells in vitro
The 1/16 cells generated by division in vitro of isolated polarized 1/8 cells
and the 1/16 cells recovered after disaggregation of 16-cell embryos display
similar features of surface organization (Ziomek & Johnson, 1981). The present
study confirms this observation, the incidence of cells with ligand-binding poles
being 58 % after disaggregation from 16-cell embryos (Table 1; line 2), and 59 %
63
Restricted
,
*
N,
T
L
Hs
1 0 1 7 5
(17) (29) (8)
Disaggregation after culture in HRP
76
12
29
9
(16) (40) (12)
• T = tight localization; L = loose localization; HS = horseshoe; 2-p = 2-poIes; cap =
Disaggregation before culture in HRP
Total
cell
no.
2 1 1 3 1 1
4
(3) (2) (22) (19)
2 0
7 1 4
3
(3) (0) (10) (19)
nuclear cap; Ring = nuclear ring; Ags = aggregates
A
,
2-p Cap Ring Ags Unscoreable
Non-restricted
Staining pattern (%)
Table 2. Incidence of staining patterns in cells from 16-cell mouse embryos (70-74 h post-hCG) incubated in
HRP before or after disaggregation to single cells
m
<
O
f.
D
CO
Cytoplasmic organization in the mouse morula
X
Fig. 3. The scale bar = 30/im. Polarized 1/8 cells were cultured, and allowed to
divide, in HRP. In established 2/16 couplets (> 1 h after division), five different
combinations of relative size and fluorescent pole presence among the two cells
were identified, (a) Smaller apolar cell (nuclear cap) and larger polar cell (tight
localization), (b) Smaller polar cell (tight localization) and larger apolar cell (nuclear
cap), (c) The cells are of different sizes; both have poles and show loose localization
of HRP. (d) The cells are of similar sizes, with the right possessing a fluorescent
pole. Both show tight localization of HRP. (e) The cells are of similar sizes, and
both have fluorescent poles. Both show loose localization of HRP. (See Johnson
&Ziomek, 1981).
Fig. 4. A 1/8 cell in division has a dispersed distribution of HRP.
199
200
W. J. D. REEVE
after generation by division in vitro of isolated 1/8 cells (Table 1; line 3). Likewise, the patterns of cytoplasmic staining were in general similar for a population of 1/16 cells labelled with HRP before disaggregation of whole embryos
and for a population of 1/16 cells formed from the division in vitro of isolated
1/8 cells (Table 1; compare lines 1 and 2 with line 3). The only significant
difference was that the area of HRP localization appeared slightly more restricted in the cells of in vitro generated couplets, in which the incidence of the
tight localization pattern was greater than in cells from intact morulae (Table 1;
Fig. 1). The difference between the staining patterns shown by the two cells of
a 2/16 couplet may arise artificially during scoring since the HRP localization
patterns are directly compared in the cells of a couplet, whereas cells obtained
from embryos pulsed with HRP prior to disaggregation are scored individually,
and direct comparisons will tend to exaggerate small differences.
In all couplets, the enzyme in polar cells was localized in the cytoplasm
underlying the pole, but in apolar cells the enzyme localization could not be
related consistently to the point of contact with, or the site of HRP in, the
polar cell (Fig. 3).
(4) The two cells of a couplet stain differently
After division in vitro of a polarized 1/8 cell, the two cells of a 2/16 couplet
can usually be distinguished by the criteria of size, pattern of fluorescent ligand
binding and microvillous distribution (Johnson & Ziomek, 1981). Commonly,
the larger cell has a restricted area, or pole, of intense fluorescent ligand binding
that coincides with a defined area of dense microvilli. The smaller cell binds
ligand uniformly over its surface of sparse microvilli. A difference between the
cells of a 2/16 in vitro generated couplet can also be demonstrated by staining
for the distribution of previously ingested vesicles of HRP, and can be observed
whether division has occurred in the presence of HRP, or after removal from
HRP and restoration to control medium.
In this study, the in vitro generated 2/16 couplets were classified according
to whether they contained one or two cells with a pole of ligand binding, and
whether or not the cells of a couplet showed an obvious size difference. A
size difference between the cells was detectable in 304 (84 %) out of 361 couplets.
The relative cell sizes within a couplet affect the patterns of cytoplasmic staining;
the larger cells show a significantly greater incidence of the restricted patterns
of staining than do the smaller cells.
In couplets containing different sized cells, over 60% of the larger cells
showed restricted localization of HRP, whereas a similar pattern was shown
usually by less than 30% of the smaller cells (Figs. 3a-c; Tables 3 and 4). In
most couplets, only the larger cell was polar (Fig. 3d) and in those cases 78 %
of polar larger cells, compared with only 33 % of smaller apolar cells, had a
restricted localization of HRP staining (Table 3). Out of 146 antibody-labelled
couplets, only four couplets examined between 1 and 2 h after division
26
8
2-3 h
3-4 h
100
44
1-2 h
Totals
22
nf
60-80 min
Total no
2/16 couplets
offpr
division
rpj
78
4
(50)
(2)
(3D
(46)
(25)
8
12
21
(48)
13
(20)
6
(27)
12
Loose
(55)
Tight
A
Restricted
(25)
2
(19)
5
(18)
8
(14)
3
Others
22
1
(5)
2
(5)
1
(4)
0
(0)
Aggregates
A
Non-restricted
Polar larger cell
(25)
2
(35)
9
(14)
6
(14)
3
Tight
33
A
(13)
2
(8)
1
(18)
2
(9)
8
Loose
Restricted
A
V
(50)
4
(42)
11
(55)
24
(64)
14
Others
67
(13)
1
(15)
4
(14)
6
(14)
3
Aggregates
A
Non-restricted
Apolar smaller cell
Cytoplasmic staining pattern (%)
Table 3. Cytoplasmic staining patterns in 1/16 cells when the two cells of a couplet derived from in vitro division of a 1/8
cell could be distinguished by size, with only the larger cell showing polarized surface binding of fluorescent ligand
•§
1
o
to
©
a
©
s
S
Si
asm
46
29
4
9
7
40-60 min
60-80 min
1-2 h
2-3 h
3-4 h
(%)
63
(34)
4
(9)
2
(7)
0
(0)
0
(0)
0
(0)
(10)
4
(13)
6
Aggregates
120
A
(66)
4
(57)
3
(11)
1
(25)
1
(28)
8
(28)
13
(3D
13
(43)
(44)
(44)
0
(0)
4
(50)
(25)
4
2
(17)
(48)
1
5
(30)
(33)
14
14
(43)
(17)
15
18
7
05)
30
Others
(65)
Loose
3
(7)
7
Tight
Non-restricted
(29)
2
(22)
2
(25)
1
(24)
4
(9)
7
(24)
10
(13)
6
Tight
49
(27)
(14)
1
(3)
0
(0)
0
(0)
1
(13)
2
(5)
6
(15)
7
Loose
Restricted
19
(41)
21
(50)
30
(65)
18
(62)
3
(75)
7
(78)
3
(43)
• ~ \
134
(73)
14
(30)
9
(21)
6
(13)
3
(10)
0
(0)
0
(0)
1
(14)
-\
Aggregates
Non-restricted
'
Others
Smaller cell
* The staining patterns have been omitted for 31I couplets in which the cells of a pair could not be distinguished by size.
183
42
20-40 min
Totals
46
Total nn nf
2/16 couplets
0-20 min
division
Restricted
Larger cell
Cytoplasmic staining pattern (%)
Table 4. Cytoplasmic staining patterns in cells when couplets were not labelled with fluorescent ligand, but when
the two cells of a couplet could be distinguished by size*
to
w
w
m
3*
s
Cytoplasmic organization in the mouse morula
203
contained a smaller polar cell and a larger apolar cell (Fig. 3b). The smaller and
larger cell types in these couplets stained similarly, each group containing 3
restricted and 1 non-restricted localization patterns. Seventeen couplets that
were examined between 1 and 3 h after their formation, and in which the cells
were of different sizes, consisted of two polar cells (Fig. 3 c). Among this group,
14 of the larger polar cells had restricted and 3 had non-restricted localizations,
compared with the incidences among the 17 smaller polar cells of 12 restricted
and 5 non-restricted localizations.
When the cells of a couplet were of similar sizes, two classes of couplet
were identified, according to whether one (Fig. 3d) or both (Fig. 3e) cells
showed polarized ligand binding. For 16 couplets in which the two cells were
of similar sizes and only one showed polar binding, the polar population contained 13 restricted and 3 non-restricted localizations; the 16 apolar cells
included 10 restricted and 6 non-restricted localizations. Nine couplets contained cells of similar sizes, both of which showed polar ligand binding. Among
the 18 cells, there were 15 restricted and 3 non-restricted localizations.
The results suggest that the pattern of cytoplasmic localization of ingested
HRP is affected by cell size and the presence of a microvillous pole, but the
difference in staining between the two cells of a couplet does not depend on a
difference in their endocytotic activities. The patterns of staining were examined
after polarized 1/8 cells had been cultured in HRP for at least 3 h, and restored
to control medium just before cell division. Of the newly formed 2/16 couplets
that were examined, 30 were considered to contain both a polar and an apolar
cell, using criteria of differences in cell size and fluorescent intensity of bound
ligand. Both cells of 2 couplets showed restricted staining, and both those of
6 couplets had non-restricted patterns of staining. However, in most cases, in
22 couplets, the polar cell showed restricted staining, and the apolar cell,
non-restricted staining.
The microvillous pole of a polarized cell is re-established during the first
hour after division, when fluorescent ligand is bound uniformly (Johnson &
Ziomek, 1981). Throughout this period, the orientation of the restricted localization of HRP cannot be related to the axis of surface polarity, and therefore
2/16 couplets examined within this time of their formation were not labelled
with fluorescent ligand. Examination of couplets at later times showed that the
patterns of HRP staining did not differ whether (Table 3) or not (Table 4) cells
had been labelled by indirect immunofluorescence. The staining patterns of
HRP appear to have stabilized within about 1 h of division (Tables 3, 4).
Prior to 1 h, the distribution of HRP is more dispersed; the larger cells of
couplets have a lower incidence of the tight localization pattern, and the smaller
cells show a high incidence of the aggregate staining distribution (Table 4).
Cells undergoing division showed a low level of dispersed HRP (Fig. 4).
204
W. J. D. REEVE
DISCUSSION
There has been little examination of cytoplasmic reorganization during
preimplantation development of the mouse embryo. Although the rat embryo
has been examined extensively by transmission electron microscopy (Schlafke &
Enders, 1967; Dvorak, 1978), studies on the mouse embryo have either been
very preliminary (Calarco & Brown, 1969; Van Blerkom & Motta, 1979) or
have tended to focus on areas of cell contact (Ducibella et al. 1977). The
immunofluorescent labelling of a range of early preimplantation stages has
indicated distinct cytoplasmic localizations for several filamentous systems, but
their organization did not appear to vary with the different developmental
stages up to the morula (Lehtonen & Badley, 1980). Some information on
cellular organization has arisen from cell lineage investigations involving the
injection of marker oil drops into the cytoplasm of cleavage-stage embryos.
The eventual location of the drop in the blastocyst could often be predicted from
the site of intracellular injection (Wilson, Bolton & Cuttler, 1972; Graham &
Deussen, 1978), and any droplets that were formed by fragmentation of the
injected drop remained clustered (Wilson et al. 1972). Although the oil had
been introduced passively into the cell, and may possibly have acted as an
inert substance, these observations nonetheless suggest that some areas at least
of cytoplasm are relatively stable. In contrast, Graham & Deussen (1978)
interpreted movement of the oil droplet with respect to the cleavage furrow as
suggesting some redistribution of the cytoplasm.
The results reported here using HRP as a cytoplasmic marker do not offer
support for a rigid cytoplasmic organization. Thus, although cells of 8-cell
embryos endocytose HRP, which becomes markedly restricted to a juxtanuclear position underlying the external surface of the cell (Reeve, 1981), the
HRP-vesicle distribution becomes diffuse at division to 2/16 cells and then
becomes progressively more organized. Stable patterns of HRP distribution
are evident within one hour of division (Tables 3, 4), but are more variable
than those described previously for the 8-cell embryo (Reeve, 1981). In the
16-cell embryo the population of outside cells shows a greater incidence of
restricted staining patterns of HRP than does the population of inside cells, and
this restricted localization also underlies the fluorescent pole. Populations of
inside cells show a higher incidence of a diffuse distribution of HRP and any
localizations that are observed are not systematically related to the point of
contact with an outer cell. What might the basis for these differences be?
Firstly, although couplets degrade HRP very rapidly, and show negligible
staining within 30 min of their restoration to control medium, there is no
evidence to relate the variation in patterns of staining to cellular differences
in degradative rates. Secondly, the differences in patterns of staining of the two
populations might be due to variations in cellular apposition or access to HRP
(Hastings & Enders, 1974). This possibility can be excluded since the distinct
Cytoplasmic organization in the mouse morula
205
patterns are also present both in populations of 1/16 cells isolated from intact
embryos prior to culture in HRP (Table 2), and in cells of 2/16 couplets generated by the division in vitro of polarized 1/8 cells (Tables 3, 4). Thirdly, differences in patterns of staining between polar and apolar cells could reflect
differences in the endocytotic activities of the two cell types. For example, it is
possible that a polar cell, which has a heterogeneous surface containing a
dense microvillous region and an area of few microvilli, does not ingest HRP
uniformly over its entire surface. Thus, preferential uptake might occur at the
region of the microvillous pole, and this could cause an aggregation of HRPcontaining vesicles in the underlying cytoplasm. In contrast, apolar cells have a
uniform distribution of sparse microvilli, and endocytosis of HRP might occur
uniformly over the surface, resulting in a low level of ingested HRP over a
widespread cytoplasmic distribution. Thus, variation in cell surface features
could cause differences that are observed in the cytoplasmic organization of
HRP-treated cells. However, it seems unlikely that the difference in staining
patterns between polar and apolar cells depends on differences in endocytotic
activities of the two cell types, since of 31 polarized 1/8 cells cultured in HRP
for several hours, and restored to control medium just before cell division, 22
divided to give a population of couplets in which the polar cells showed a
greater incidence of restricted staining than did the population of apolar cells.
This result makes it seem likely therefore that, at division of 1/8 cells, the larger
polar cells generated must inherit either more endocytosed vesicles or some
features responsible for the tighter aggregation of vesicles under the pole in an
area of the cytoplasm known in other cell types to be occupied by the Golgi
complex (Steinman, Silver, & Cohn, 1974; Piasek & Thyberg, 1979).
In conclusion, the evidence from this study, taken with that from other studies
referred to above, suggests that although there may be considerable cytoplasmic
disturbance during cleavage divisions, some elements of stability within the
cytoplasm may occur. The data are not therefore inconsistent with a model
for blastocyst development that ascribes an important role for differential
inheritance of various cellular features at the 8- to 16-cell division (Johnson
et al. 1981), although they do not in themselves constitute adequate proof of
such a model.
Finally, although the population of polar outer cells shows a greater incidence
of restricted localization of HRP than does the population of inner apolar cells,
the differences between the two populations are not absolute and thus, unlike
the differences in surface phenotype (Handyside, 1981; Johnson & Ziomek,
1981), cannot be diagnostic for position. It is possible, but improbable, that
two discrete populations exist in situ, but that the sharp differences in HRP
localization become blurred during cell analysis as a result of misscoring or
technical manipulations. For example, some inaccuracies will arise from
misassignment of cells to one of the seven categories of staining owing to
problems of interpreting 3-dimensional distributions in a 2-dimensional
206
W. J. D. REEVE
analysis. It is unlikely that the patterns represent an artifact induced during
cell preparation since observations on intact stained embryos, although not
easy to make, indicate that the range of patterns among inside and outside cells
in situ is similar to that observed in isolated cells. Moreover, among isolated
cells the range of patterns does not vary with the method of cell isolation and
the time of labelling. There is heterogeneity within each population, and it
seems probable that there is a genuine overlap of HRP localization patterns
between each population. It is not clear whether this heterogeneity is of developmental significance.
I wish to thank Drs C. A. Ziomek, H. P. M. Pratt and M. H. Johnson for constructive
criticism, and Gin Flach, Mike Parr and John Bashford for technical help. The work was
supported by grants from the Ford Foundation, the Cancer Research Campaign and the
Medical Research Council to Dr. M. H. Johnson, and from the Medical Research Council
and the Cambridge Philosophical Society to the author.
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{Received 1 May 1981, revised 20 June 1981)