J. Embryol. exp. Morph. Vol. 70, pp. 261-278, 1982
Printed in Great Britain © Company of Biologists Limited 1982
261
Cosegregation of monoclonal antibody
reactivity and cell behaviour in the mouse
preimplantation embryo
ByBEVERLEY J. RANDLE1
From the Department of Zoology, South Parks Road, Oxford
SUMMARY
Expression of an antigen, recognized by a monoclonal antibody raised against PCI 3
embryonal carcinoma, is described in mouse preimplantation embryogenesis. The antigen is
found in the cytoplasm of ovulated ova and is first noted on the cell surface of the 1-cell
embryo 20 h post-ovulation. Surface labelling of blastomeres is uniform until the 8-cell stage
when antigen expression becomes polarized along the radial axis of the embryo. Two major
populations of blastomeres are distinguishable on division to the 16-cell morula. Dissociation
of morulae in calcium-free medium yields large, polar, antigen-positive cells and small
apolar cells with reduced levels of detectable antigen. A third, minor population of small,
antigen-negative cells is also found in vivo. Large and small blastomeres differ in their ability
to relocate within the embryo when aggregated with intact 16-cell-stage embryos. The small
blastomeres of the 16-cell morula contribute significantly to the inner cell mass while the
large antigen-positive cells are found only in the trophectoderm.
INTRODUCTION
The formation of new tissue in embryos is thought to arise as a result of cell
interactions. The cell surface is believed to play an important role in the transmission and transduction of developmental signals and in the appearance of
differentiated function, (for recent reviews see Subtelny & Wessels, 1980). The
preparation of monoclonal antibodies by fusion of splenocytes of immune
animals with established myeloma cell lines (Kohler & Milstein, 1975) has
provided reagents for the dissection of structure and function of the cell surface.
It is now possible to begin to investigate the relationship between cell phenotype,
described by collections of reagents recognizing single antigenic determinants,
and the ability of a cell to divide and differentiate in a developing system.
Blastomeres of the early 8-cell-stage mouse embryo have similar cell surfaces
with distinct surface characters segregating to different cells at division to the
16-cell stage (Ziomek & Johnson, 1981). This process has been studied using a
novel monoclonal antibody, clone 2C5, raised against mouse embryonal
carcinoma. Antibody reactivity is first noted in the ovum cytoplasm and later
1
Author's present address: Reproductive Immunology Group, Bristol Royal Infirmary,
Marlborough Street, Bristol BS2 8HW, U.K.
262
B. J. RANDLE
(e)
Fig. 1. Indirect immunofluorescence assay of 2C5 monoclonal antibody on live
preimplantation embryos. Embryos at (a) 16 h, (b) 20 h, (c) 36 h, (d) 58 h, (e) 72 h,
and (/) 88 h post ovulation and (g) inner cell mass, isolated by immunosurgery,
88 h post-ovulation are shown. Phase contrast optics and ultraviolet illumination.
Scale bar («)-(/) 40/im, (g) 20 /nm.
in development, 2C5 antigen is detected on the cell surface of preimplantation
mouse embryos. At the 16-cell stage, a differentiation related to antibody
binding is apparent. The reaction of the blastomeres at the 16-cell stage with
monoclonal antibody 2C5 is shown to distinguish cells which also differ in their
behaviour on aggregation with preimplantation embryos.
MATERIALS AND METHODS
Preparation of monoclonal antibody cell line 2C5
Cell line 2C5 was isolated from a fusion experiment between mouse myeloma
P3-NSl/lAg4-l, subsequently NS1, (Kohler, Howe & Milstein 1976) and
splenocytes from a female C3H mouse immunized with whole cells of PCI3
embryonal carcinoma, clone 5 (Bernstine, Hooper, Grandchamp & Ephrussi,
1973; Adamson, Gaunt & Graham, 1979). Two immunizations of 5 x 106 PCI3
Monoclonal antibody reactivity to early mouse embryos
263
Fig. 2. Indfrect immunofluorescence assay of 2C5 monoclonal antibody on sections
of ovaries and preimplantation embryos, (a) and (b) show ova with unfused and
fused follicular antra respectively. Embryos at (c) 16 h, (d) 24 h, (e) 8-cell stage,
58 h, (/) 8-cell stage, 66 h, (g) 16-cell stage, 72 h and (h) blastocyst, 88 h. Phasecontrast optics and ultra-violet illumination. Scale bar 40 /im.
clone 5 cells in Freund's complete Adjuvant (Gibco Ltd., New York) were given
intramuscularly at an interval of two weeks. A month later, a final injection of
5 x 106 PC13 clone 5 cells in alpha medium (Stanners, Eliceiri & Green, 1971;
Flow Laboratories, Irvine) was given intravenously. After 4 days, the mouse was
killed and 4 x 107 spleen cells, purified on Ficoll Paque (Pharmacia Fine
Chemicals, London) were mixed with 4 x 106 NS1 cells. The cells were washed
three times in alpha medium and resuspended in 150 /A 50 % (w/v) polyethyleneglycol 1500 (B.D.H. Ltd., Poole) in alpha medium for 75 s at room temperature.
The suspension was then diluted to 10 ml over the following 5 min with alpha
HAT medium, consisting of alpha medium supplemented with 10% (v/v) heat
inactivated foetal calf serum (Flow Laboratories) and hypoxanthine, methotrexate and thymidine (Sigma, London; Littlefield, 1964). Cells were then
pelleted at 650 rpm for 2 min and finally plated out into four 24-well Linbro
multiwell plates (Flow Laboratories) in 1-5 ml alpha HAT medium per well.
Cultures were fed seven days post fusion with fresh alpha HAT medium and
then fed at three-day intervals with testing of culture supernatants for production of antibody 19 days post fusion.
Production of antibody to the cell surface of PC13 clone 5 was detected by
264
B. J. RANDLE
Table 1. Antigen expression on dissociated blastomeres
% cells
Distributioni of 2C5 antibody*
Embryonic: h post
No. of
stage ovulation ] blastomeres Ring 3/4 1/2 Polar Non-flf Ring Non-ring Non-fl
A
A
2-cell
4-cell
8-cell
8-cell
8-cell
16-cell
26
38
58
62
66
72
32
38
90
132
126
208
32
38
82
96
16
87
_
—
1
8
11
2
_
—
5
13
32
23
_
—
2
15
67
54
_
—
—
—
—
42
100
100
91
73
12
42
9
27
88
38
—
—
—
20
* Detected with rabbit anti-mouse immunoglobulin G antibody conjugated to fluorescein.
t Non-fluorescent.
radioassay using methods of Jensenius & Williams (1974), with 2x 105 whole
PCI3 clone 5 cells as a target for each sample assayed.
Cells from positive well 2C5 were subcultured and cloned in 0-325 % (w/v)
soft agarose (Miles Biochemicals Ltd., Stoke Poges) in alpha HAT medium
overlaying 0-625 % (w/v) soft agarose in alpha HAT medium.
Clones 2C5 cell lines were then maintained in alpha HAT medium and
supernatants from cultures were stored at - 20 °C until use.
2C5 antibody is a mouse immunoglobulin M as indicated by double immunodiffusion of culture supernatant against mouse heavy chain specific
antisera (Miles Biochemicals Ltd; Method of Ouchterlony, 1958). 2C5 cloned
cell lines produce up at 15 jug antibody per ml medium as shown by staining of
10% polyacrylamide gels (Laemmli, 1970) of culture supernatants with Coomassie Blue.
Supply of ova and preimplantation embryos
Embryos were from natural matings of female 129J/Sv mice with ¥x (CBA x
C57) males of proven fertility. Males were either caged overnight with females
or introduced to female stock for one hour in the morning - the latter procedure
giving an exact time of mating. Females were inspected for copulation plugs
and the day of plug was the first day of pregnancy.
Zygotes were dissected from the ampullae on the first day of pregnancy.
2- and 4-cell stages were recovered in the second day of pregnancy from the
oviduct. 8-cell embryos and morulae were removed from the oviduct on the
third day of pregnancy. Blastocysts were flushed from the uterus on the fourth
day of pregnancy.
Zonae pellucidae were removed by brief incubation of embryos in acid tyrodes
solution (Nicolson, Yanagimachi & Yanagimachi, 1975).
Inner cell masses were isolated from blastocysts by immunosurgery (Solter &
Knowles, 1975), using 2C5 supernatants. Blastocysts were incubated for 30 min
Monoclonal antibody reactivity to early mouse embryos
265
Fig. 3. Indirect immunofluorescence assay of 2C5 monoclonal antibody on dissociated blastomeres of preimplantation embryos, (a) one blastomere of 2-cell
stage, 26 h, (b) one blastomere of 4-cell stage, 42 h, (c) 1/8 cell, ring stain 58 h,
id) 1/8 cell, 3/4 stain, 62 h, (<?) 1/8 cell 1/2 stain, 62 h, (/) 1/8 cell, polar stain,
66 h, (g) 1/16 cell, ring stain, 72 h, (h) 1/16 cell, bright cell ring stain, 72 h, (/) 1/16
cell, polar stain, 72 h, (;) 1/16 cell, non fluorescent, 72 h, (k) 1/8 cell dividing to
2/16 pair, in vitro, 70 h, (/) 2/16 pair, bright and dim, in vitro 72 h, (w) 2/16 pair,
equal fluorescence and equal size in vitro, 72 h. Phase contrast and ultraviolet
illumination. Scale bar 40/mi.
at room temperature in 2C5 medium, washed in phosphate-buffered saline
(Dulbecco & Vogt, 1954) and then placed in guinea-pig serum for 5 min. The
lysed trophectoderm cells were then removed by gentle pipetting.
Dissociation of embryo blastomeres
Preimplantation stages were dissociated into individual blastomeres by use
of calcium-free M16 medium (Whittingham, 1971) and a series of micropipettes
pulled by hand to have internal diameters varying from 100/^m to 25 ju,m. All
pipettes were polished using a de Fonbrune Microforge (C. Beaudouin, Paris).
Embryos were incubated in microdrops of M16 medium with 0-6% (w/v)
bovine serum albumen (BSA) (Armour Pharmaceuticals) lacking calcium and
magnesium, under paraffin oil (Boots Pharmaceuticals, Ltd.), for 5-20 min at
37 °C in a humidified atmosphere of 5 % (v/v) carbon dioxide in air. The
blastomeres were then gently pipetted apart and placed in M16 medium with
0-4% (w/v) BSA to recover before use.
n
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3
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. Number of cells
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z
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o
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Number of cells
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Number of cells
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Monoclonal antibody reactivity to early mouse embryos
267
Table 2. Antigen expression of 1/8 blastomeres divided to 2/16 pairs
Fluorescence pattern related
to cell size in each pair
Bright, large/Dim, small
Equal size, both bright
Equal size, both dim
No. of pairs
% total number
of pairs
75
2
11
85
2
13
Total number of 2/16 pairs tested = 88 pairs.
Blastomeres isolated from 8-cell embryos are termed 1/8 cells, while blastomeres from 16-cell embryos are termed 1/16 cells. 1/8 blastomeres divide to give
2/16 daughter pairs.
Culture of embryos and isolated blastomeres
Embryos and blastomeres were cultured on tissue culture dishes in preequilibrated microdrops of Ml6 medium covered with paraffin oil and maintained in a humidified atmosphere of 5 % (v/v) carbon dioxide in air at 37 °C.
Fixation and sectioning of embryos for immunofiuorescence
Embryos were washed briefly in phosphate-buffered saline and then placed in
Bouin's fixative for 5 min. 2C5 antigen is stable in Bouin's fixative as indicated
by comparison of absorption of 2C5 antibody by fixed and unfixed PC 13
embryonal carcinoma in a quantitative assay (unpublished data; for methods
see Gaunt (1982)). 2C5 antibody immunofluorescence on sections of 7-day
embryos was compared with live 7-day embryonic tissue isolated by dissection.
2C5 antigen is expressed on embryonic endoderm but 2C5 antibody does not
react with 7-day embryonic ectoderm (unpublished data). The distribution of
2C5 antigen is identical in both tissue sections and embryos in which the cell
layers are dissected apart. Tissue sections therefore appear to be a reliable guide
to distribution of antigen on living cells.
Fixed embryos were placed in molten 1 % (w/v) agar in 0-9% (w/v) saline at
40 °C. The agar was allowed to set and was cut into blocks. Blocks were
processed through alcohols and xylene and embedded into paraffin wax.
Blocks were then sectioned at 8 [im.
Sections for immunofluorescence were rehydrated through xylenes and
alcohols, washed for 20 minutes in running water and then rinsed in phosphatebuffered saline ready for use.
Fig. 4. Indirect immunofluorescence of blastomeres isolated from the 16-cell embryo.
Each histogram represents one embryo. Cell diameter in /tm is plotted against
number of cells with a non polar, polar and non-fluorescent pattern of antibody
binding.
268
B. J. RANDLE
Table 3. Incorporation of large 1/16 cells into morulae cultured to early
blastocyst
No. of labelled cells
No. of unlabelled cells
A
Embryo
1
2
3
4
5
6
7
8
9
10
11
'inside'
A
'outside'
'inside'
'outside'
22
—
23
—
20
—
19
—
30
—
25
—
21
—
21
—
21
—
33
—
27
—
'Inside' indicates a cell with no contact with medium.
'Outside' indicates a cell with contact with medium.
5
6
5
5
8
6
12
8
11
6
7
1
1 (unincorporated)
1
1
1
1
1
1
1
2
1
Indirect immunofluorescence of live and fixed, sectioned preimplantation material
2C5 supernatants were supplemented to 2 rr»M Hepes pH 7-4 and 0-02 % (w/v)
sodium azide.
Live material was incubated for 30 min in 50 fi\ drops of 2C5 reagent on a
petri dish placed over an ice bath. Embryos were then washed in wash buffer
(phosphate-buffered saline, 0-5% (w/v) BSA, 0-02% (w/v) sodium azide, 2 mM
Hepes pH 7-4). Embryos were placed in 50/d drops of rabbit anti-mouse
immunoglobulin G antibody conjugated to fluorescein (Miles Biochemicals
Ltd.) diluted 1/16 in wash buffer. After a 30 min incubation over an ice bath the
embryos were washed in wash buffer. Embryos were placed in a microdrop of
wash buffer on a haemocytometer slide and covered with a quartz glass coverslip. The preparation was viewed using a Zeiss RA microscope with a type IV
epifluorescence condenser attachment and phase contrast optics.
Sections of embryos and ova were treated in situ with 20 pt\ of 2C5 reagent
and 4 °C for 30 min. After washing, 20 pt\ of rabbit anti-mouse immunoglobulin
G antibody at 1/16 dilution were placed over the material for 30 min at 4 °C.
Sections were then washed three times with wash buffer and covered with a
quartz coverslip for examination.
Immunofluorescence assays of 2C5 antibody binding were controlled using
medium from cultures of 1B3 monoclonal antibody, a gift from Dr S. J. Gaunt.
1B3 antibody is a mouse immunoglobulin M recognizing a 28 K daltonpolypeptide on post-pachytene spermatocytes (Gaunt, 1982). 1B3 antibody produces
5-10 fig antibody per ml medium as indicated by staining with Coomassie Blue
of polyacrylamide gels of culture supernatants.
Monoclonal antibody reactivity to early mouse embryos
(a)
269
(b)
Fig. 5. Photomicrographs of autoradiographs of sections of early blastocysts
cultured from aggregates of a 16-cell-stage embryo and one 1/16 blastomere.
Aggregation of (a) small 1/16 cell, (embryo one, Table 4), (b) large 1/16 cell,
(embryo two, Table 3) unincorporated and (c) large 1/16 cell, (embryo five, Table 3).
The plane of sectioning of (a) is through the inner cell mass. Scale bar 40 fim.
Photographs were taken with Kodak Technical Pan film 2415 with an exposure
time of 90 s for immunofluorescence.
Labelling ofS-cell-stage embryos
Embryos were labelled with [3H]thymidine using conditions described by
Kelly & Rossant (1976) where labelling did not affect subsequent growth and
differentiation. Embryos were dissected from the oviduct 58 h postovulation
and zonae pellucidae were removed by incubation in acid tyrodes solution.
Embryos were washed in pre-equilibrated Ml6 medium with 0-4% (w/v) BSA
and then incubated in microdrops of M16 medium with 0-4% (w/v) BSA
supplemented with [3H]thymidine at a concentration of 0-25 /tCi per ml
(Specific activity 44Ci/mMol, the Radiochemical Centre, Amersham). Embryos
were cultured in label for l-|h and then washed in fresh M16 medium with
0-4% (w/v) BSA. Finally, embryos were cultured in Ml6 medium with 0-4%
(w/v) BSA until use at 72 h postovulation.
if
270
B. J. RANDLE
Table 4. Incorporation of small 1/16 cells into morulae cultured to early
blastocyst
No. of labelled cells
No. of unlabelled cells
A
Embryo
1
2
3
4
5
6
7
8
9
'inside'
6
7
5
7
8
8
6
7
5
A
'outside'
'inside'
'outside'
34
19
23
22
21
21
19
24
26
2
1
—
1
2
1
—
1
2
_
—
1
1
—
—
1 (unincorporated)
—
—
<Inside' indicates a cell with no contact to medium.
Outside' indicates a cell with contact to medium.
Aggregation of 1/16 blastomeres ami host-labelled embryos
1/16 blastomeres for aggregation were prepared as follows. 8-cell embryos,
62 h post mating, were dissociated in calcium-free M16 medium. 1/8 blastomeres were then cultured individually overnight to the equivalent of 72 h
postfertilization. During this culture period, 80% 1/8 blastomeres divided to
2/16 cell pairs. 1/16 blastomeres were prepared by disaggregation of 2/16 cell
pairs in calcium-free Ml6 medium. 2/16 pairs were disaggregated individually
and the large and small cells from each couplet placed in separate microdrops.
Single 1/16 blastomeres, sorted into large and small size populations, were
placed next to labelled 16-cell embryos. 72 h postovulation, cultured individually
in microdrops. After 30 min each embryo was inspected to ensure the introduced
blastomere had stuck. The chimaeric embryos were cultured for 8 h to early
blastocoel formation and then fixed for autoradiography.
Fixation, sectioning and autoradiography of blastomere embryo aggregates
Embryos were fixed, embedded and sectioned as for immunofluorescence.
Sections for autoradiography were rehydrated and then washed in two changes
of 5 % (w/v) trichloracetic acid at 4 °C for 30 min. The sections were washed
for 1 h under running tap water, rinsed in distilled water and dipped in Ilford
K2 emulsion (Rodgers, 1979).
Slides were exposed for two weeks at 4 °C and then developed with Kodak
D19 developer and fixed with Kodafix Losung. Slides were then stained with
Mayer's Haemalum and light green, dehydrated and mounted for examination.
Distribution of nuclei and presence of grains, indicating labelling with [3H]
thymidine, was noted for each section.
Monoclonal antibody reactivity to early mouse embryos
271
RESULTS
The results are divided into three sections. 2C5 antigen detection is described
for the cell surface and cytoplasm of preimplantation embryos. Secondly, the
origin of two populations of blastomeres in the 16-cell embryo with differing
localizations of 2C5 antigen, is investigated by following division of single
blastomeres isolated from 8-cell embryos. Finally, the functional properties of
two populations of blastomeres of the 16-cell stage are tested by an aggregation
experiment.
1. 2C5 antigen detection
(i) on the cell surface of intact ova and preimplantation embryos. Ova and
embryos were assayed for the presence of antigen by indirect immunofluorescence. At all stages tested, a minimum of ten embryos from at least two litters
were assayed. The results are illustrated in Fig. 1. The antigen is first detected
20 h postovulation, Fig. 1 a, b, and is expressed on the surface of 2-, 4- and 8-cell
stages, Fig. \c, d. The external surfaces of the 16-cell stage, Fig. 1 e, and the
trophectoderm Fig. I/react with the monoclonal antibody. The inner cell mass
does not express 2C5 antigen, Fig. lg. Unfertilized ova react with the monoclonal antibody 20 h postovulation.
Ten zygotes were removed from the reproductive tract 4 h postfertilization
and cultured in vitro to the late 2-cell stage. 2C5 antigen was detected on the
surface of all cultured 2-cell stage embryos illustrating initiation of antigen
expression in vitro.
(ii) in the cytoplasm of ova and preimplantation embryos. Sections of ova and
preimplantation embryos were assayed for presence of 2C5 antigen by indirect
immunofluorescence. Ten embryos, derived from at least two litters, were
assayed at each stage.
2C5 antibody reacts with the cytoplasm of ova with fused antra swollen with
follicular fluid, Fig. 2b but not with the cells of the cumulus oophorus or with
immature or atretic follicles, Fig. 2a.
Distribution of 2C5 antigen is uniform in the cytoplasm of ova, Fig. 2a, b,
zygotes Fig. 2 c and 2-cell embryos, Fig. 2 b. In the early 8-cell stages, Fig. 2e,
the monoclonal antibody reacts with an area under the cell surface of contacts
between blastomeres. At compaction, the apical cytoplasm is heavily labelled
and reactivity is reduced in the regions of interblastomere contact, Fig. 2 /
Cells are found internally in the 16-cell stage which do not label with monoclonal antibody in the cytoplasm, Fig. 2g. In the blastocyst, inner cell mass
cytoplasm is antigen negative and 2C5 antigen is detected in the trophectoderm
cell cytoplasm, Fig. 2h.
(iii) on the cell surface of dissociated embryos. Embryos from the 2-cell stage
to the 16-cell stage were dissociated using calcium-free medium and the cell
surface stained with monoclonal antibody detected by immunofluorescence.
272
B. J. RANDLE
The results are shown in Table 1 and Fig. 3. The distribution of antibody
reactivity varied from a ring fluorescence e.g. Fig. 3 a to a highly polar localization, e.g. Fig. 3/. Intermediate staining patterns in which threequarters and
half the cell surface was positive were noted, Fig. 3d, e respectively.
Initially antigen distribution is uniform over the surface of 2-, Fig. 3 a, 4Fig. 3 b and early 8-cell blastomeres, Fig. 3 c. In compacting 8 cell stages,
Fig. 3 c-f an increasing percentage of blastomeres with a polar concentration
of antigen is detected. At 66 h postovulation 88% of all 1/8 blastomeres are
polarized, Fig. 3 / At the 16-cell stage 38% of blastomeres are polarized in
distribution of 2C5 antigen, Fig. 3/, 42% show ring fluorescence, Fig. 3g, h,
and 20% of all blastomeres do not stain with the monoclonal antibody, Fig. 3j.
Ratios of polar and non-polar cells may result from non-random cell loss
during disaggregation of 16-cell embryos. Consequently, all 16 cells from single
16-cell embryos were assayed for 2C5 antigen. On five occasions, all cells from
individual 16-cell embryos were scored for antibody binding. Results are shown
in Fig. 4. The number of cells is plotted against diameter of the cells for each
class of non-polar, polar and non-fluorescent score. There is an increased ratio
of non-polar to polar cells when all cells from 16-cell-stage embryos are scored
as compared with a random population of 1/16 cells. The percentage of nonfluorescent cells is reduced from 20% in the random 1/16 cell population to
approximately 10% when all cells from 16-cell embryos are scored for 2C5
antibody reactivity.
2. Antigen expression of 1/8 blastomeres cultured to 2/16 daughter pairs
1/8 blastomeres were isolated from semicompacted embryos, 62 h postmating. The blastomeres were cultured in microdrops and tested by indirect
immunofluorescence for 2C5 antigen expression at 1 h postdivision. Three types
of staining related to cell size are noted. See Fig. 3 and Table 2. In 75/88 pairs of
2/16 cells, the bright, antigen-positive cells are always the larger cells of the
daughter pairs, Fig. 3 1. Culture of 20 pairs for 4 h postdivision indicated that
large, antigen-positive cells become polarized in antigen distribution. In
contrast, the smaller, dimmer cells are not polarized in these pairs. No nonfluorescent cells are noted in contrast to the observations on sectioned and
freshly dissociated 16-cell-stage embryos. 15% of all pairs consisted of daughter
cells of the same size. In all cases, both cells stained with monoclonal antibody
equally, both blastomeres being bright or dim, Fig. 3 m.
Fig. 3k shows a 1/8 cell in cytokinesis. 2C5 antibody reactivity indicates that
there is differential inheritance of 2C5 antigen on division of a 1/8 cell to a 2/16
daughter pair.
3. Incorporation of large and small 1/16 blastomeres into the 16-cell embryo
The ability of single blastomeres from the two major populations of the
16-cell stage to relocate in the embryo was tested by aggregation to labelled 16cell embryos.
Monoclonal antibody reactivity to early mouse embryos
273
Twelve pairs of 2/16 blastomeres, each consisting of one large and one small
cell, were dissociated in calcium-free medium and sorted into large and small
cells. Single cells were then aggregated with 16-cell embryos labelled with [3H]
thymidine. The aggregates were cultured for 8 h to the start of blastocoele
formation. Embryos were fixed, sectioned and autoradiographed to determine
the final position of the unlabelled blastomeres. Five labelled 16-cell-stage
embryos were cultured to determine whether the manipulation of aggregation
had retarded the development of the embryos. Control 16-cell embryos had an
average of 33 ± 3 labelled nuclei after culture. All nuclei in control embryos were
labelled.
Incorporation of large 1/16 blastomeres into 16-cell embryos is detailed in
Table 3. Embryos consisted of an average of 32 cells. In all cases the unlabelled
blastomere, introduced by aggregation was found to occupy'an 'outside'
position, with the cell membrane in contact with external medium. Fig. 5c
shows a section through embryo five (Table 3). The unlabelled cell has contributed to the trophectoderm layer of the early blastocyst.
/
Small cells gave a different pattern of incorporation into morulae, as indicated
in Table 4. 75% of the small blastomeres contributed to the inside of the
blastocyst alone. One blastomere was found in the outer, trophectodermal layer
and one blastomere divided to give daughter cells contributing to both the
trophectoderm and inner cell mass. An example of the distribution of the
unlabelled blastomeres in the labelled blastocyst aggregates is shown in Fig. 5 a.
Embryos consisted of an average of 34 cells and in common with the aggregations of unlabelled large blastomeres, embryonic development had not been
retarded when compared to labelled control embryos.
Embryo two, (Table 3) and embryo seven (Table 4) failed to incorporate the
introduced unlabelled blastomere. A section through embryo two is shown in
Fig. 5 b.
DISCUSSION
Monoclonal antibody 2C5, raised to PCI3 embryonal carcinoma, cross reacts
with a cell surface determinant of mouse preimplantation embryos. 2C5 antigen
is first expressed in the cytoplasm of mature ova in the ovary and is later found
on the cell surface of the zygote 20 h postovulation. Expression at the cell
surface occurs in vitro suggesting that 2C5 antigen is not absorbed from the
reproductive tract. The antigen is, however, present on the surface of oviducal
luteal cells at ovulation and on the cell surface of the uterine epithelium at
implantation (unpublished data).
Blastomere cell surfaces react with 2C5 monoclonal antibody from 20 h to
72 h postovulation. Three populations of blastomeres are found in the 16-cell
embryo with cells having a high or low level of antibody reactivity or lacking
detectable antigen expression. In the blastocyst, the trophectoderm alone is
antigen positive.
274
B. J. RANDLE
The antigenic site detected by 2C5 monoclonal antibody is distinct from the
carbohydrate reactivity of SSEA1 antibody (Gooi et al. 1981) and the Forssmann antigen recognized by Ml/22.25 (Willison and Stern, 1978). SSEA1 is
first detected on some of the blastomeres of the 4-cell embryo and is expressed
on the inner cell mass (Solter & Knowles, 1978). In contrast, the Forssmann
specificity is found in late morulae and blastocysts (Willison & Stern, 1978).
2C5 antigen distribution resembles the reactivity of ECMA2 and ECMA3
monoclonal antibodies (Kemler, Morello & Jacob, 1979), however, 2C5 antibody does not react with sperm (unpublished data) or the inner cell mass. Three
monoclonal antibodies are reported to be trophectoderm specific. TR0MA1
and TROMA2, (Brulet, Babinet, Kemler & Jacob, 1980) recognize a prekeratin
specificity and a kappa IgM, raised to F9 embryonal carcinoma (Edwards,
Foster & Mcllhinney, 1980) reacts with an unknown specificity in the preimplantation mouse trophectoderm. It is concluded that 2C5 monoclonal
antibody reacts with previously undescribed antigen of preimplantation mouse
embryos.
2C5 antigen becomes localized to the apical cytoplasm of 8-cell embryo
blastomeres at compaction. Similar cytoplasmic regionalization has been
reported by Reeve (1981) in rat and mouse embryos. Organelles stained
with acridine orange or toluidine blue were found to be arranged in columns
between the nucleus and embryo periphery in the rat 8-cell stage. In the mouse
8-cell embryo, ingestion of horseradish peroxidase was localized to the apical
cytoplasm. In contrast, Mulnard & Huygens (1978) found activity of nonspecific alkaline phosphatase in cytoplasm beneath the inter-blastomeric contacts of the 4-cell mouse embryo.
Dissociation of preimplantation embryos to isolate blastomeres reveals that
2C5 antigen distribution is polarized on the cell surface at compaction. Blastomeres, stained with 2C5 monoclonal antibody have uniform ring fluorescence
at the 2-, 4- and early 8-cell stages. Late 8-cell embryos have a polar distribution
of antigen. Similar regionalization of cell surface determinants has been noted
using fluorescently labelled concanavalin A (Con A), peanut agglutinin, and
rabbit anti-mouse species antigens (Handyside, 1980; Ziomek & Johnson, 1980).
Handyside (1980) found that 77% of blastomeres at compaction show a polarization of surface label.
Compaction is also accompanied by formation of microvillar poles (Ducibella,
Ukena, Karnovsky & Anderson, 1977; Reeve & Ziomek, 1981). Microtubules
and mitochondria localize beneath apposed cell membranes and microvilli
become reorganized to the apical region of compacting blastomeres. The
polarization of 2C5 antibody reactivity occurs at the onset of microviliar pole
formation. It is probable that 2C5 antigen concentrates at the pole of blastomeres as a result of concentration of membrane into microvilli at compaction.
Ziomek & Johnson (1981) followed division of isolated blastomeres of the 8cell embryo (1/8) to division to daughter pair (2/16). Polarity was assessed by
Monoclonal antibody reactivity to early mouse embryos
275
fluorescently labelled Con A. Two populations of blastomeres were created on
division. 82% of 1/8 blastomeres divided to give a pair of cells where one
blastomere inherited the lectin-labelled pole. The remaining cells divided
through the pole resulting in a polar 2/16 pair with the pole continuous through
the midbody. Handyside (1980) also reported the appearance of two blastomere
populations at the 16-cell stage. 53% of blastomeres of the 16-cell embryo
have a pole as revealed by fluorescent ligand binding.
Observations with 2C5 monoclonal antibody indicate that the 16-cell-stage
embryo consists of two major populations of cells. On division to the 16-cell
stage, the antigen pole formed at the 8-cell stage is inherited by one of the
daughter cells. The cell inheriting the pole is larger than the blastomere which
expresses lower levels of 2C5 antigen in a uniform distribution.
The ratio of polar to non-polar blastomeres detected by 2C5 antibody in the
16-cell embryo is very low in comparison to findings with fluorescently labelled
Con A. Johnson and Ziomek (1981) report a ratio of nine polar to seven nonpolar cells in the 16-cell embryo labelled with Con A. In the present study,
embryos are scored at 72 h postcoitum and it is possible that the number of
polar cells has been understated, the polarity not being generated until 2-3 h
later. This is supported by the finding that large bright 1/16 cells become polar
on culture for 4 h after isolation from division of 1/8 cells to 2/16 pairs.
A third population of cells was noted in dissociated 16-cell embryos and
includes 20% of all isolated 16-cell blastomeres (1/16) tested for 2C5 monoclonal antibody reactivity. The blastomeres do not express 2C5 antigen on the
cell surface. No equivalent population of cells was noted in experiments
dividing 1/8 cells to 2/16 pairs. 13% of blastomeres did however divide to give
daughter pairs of equal size and low levels of antibody reactivity. This in vitro
population may be equivalent to the antigen-negative blastomeres seen in vivo.
The significance of this population is unclear. The mechanism of removal of 2C5
antigen from the cell surface is unknown. The antigen may be shed from the cell
surface or internalized to the cell cytoplasm and rapidly destroyed. Both
mechanisms may be interrupted in vitro.
The antigen-negative blastomeres of 16-cell embryos may represent the first
appearance of inner cell mass precursor cells. 2C5 antigen cannot be detected
on inner cell mass cells. Sectioning of 16-cell embryos and consequent staining
with 2C5 antibody suggests the presence of cells inside the embryo which do
not express 2C5 antigen on their cytoplasm. Results of Garner & McLaren
(1974) indicate there is little cell mixing in preimplantation embryogenesis. It
is therefore suggested that blastomeres with very low levels of 2C5 antibody
reactivity are in a position within the 16-cell embryo where they may give rise
to the inner cell mass.
The properties of the two major populations of blastomeres of the 16-cell
morula were investigated by an aggregation experiment. 1/8 blastomeres were
cultured and on division, large and small blastomeres were isolated from 2/16
276
B. J. RANDLE
cell pairs where daughter cells were differing in size. The blastomeres were then
assayed for their ability to participate in the development of the 16-cell embryo.
Sectioning of blastocysts revealed that the large, antigen-positive 1/16 cells
contributed to the outside layer of the early blastocyst while 75 % of the small
1/16 blastomeres, with reduced levels of antigen expression, were incorporated
into the inner cell mass. This finding suggests that the ability of blastomeres of
the 16-cell stage to relocate in the 16-cell embryo differs and is related to the cell
surface phenotype identified by binding of 2C5 antibody. Small 1/16 blastomeres, placed on the outside of 16-cell embryos are able to relocate to an internal
position whilst large 1/16 2C5 antigen-positive blastomeres contribute to the
external, trophectodermal layer.
Johnson & Ziomek (1981) have proposed that two distinct cell lineages are
founded in the mouse morula. The lineages are associated with the inheritance
of microvillar poles on division of 8-cell-stage blastomeres. The two populations
of cells generated on division differ in adhesivity, surface phenotype, size and
position in the mouse morula (Ziomek & Johnson, 1981). Ziomek & Johnson
(1982) also present evidence that the developmental fate of blastomeres is
partially restricted in the early 16-cell embryo. Using a fluorescein isothiocyanate
marker, large and small 1/16 blastomeres were labelled and then aggregated to
fifteen age-matched unlabelled 1/16 cells. The aggregates were cultured and the
fluorescent-labelled cells identified. Labelled large 1/16 blastomeres contributed
mainly to the trophectoderm when placed on the outside of aggregates and gave
rise to at least one trophectoderm cell when placed inside aggregates. In contrast,
labelled small 1/16 cells placed inside gave rise to predominantly inner cell
mass and when placed outside generated cells in the inner cell mass alone, in the
trophectoderm alone or in both tissues. Ziomek & Johnson (1982) conclude that
the fate of large 1/16 blastomeres is related to surface phenotype while that of
small 1/16 blastomeres is influenced by their position within the morula.
Experiments of Rossant & Vijh (1980) suggest that all cells of the early
mouse embryo are developmentally labile until blastulation. Single 1/16
blastomeres removed from the outisde of 16-cell embryos were aggregated to
8-cell-stage embryos. 3 3 % of the embryos resulting from aggregation had
progeny of the donor blastomere in both the trophectoderm and inner cell mass.
The aggregation was however performed with cells of earlier stage embryos than
the present study. The extra 12 h in a 'developmentally foreign' environment
may have been sufficient to permit a change in behaviour of 1/16 outside cells
on aggregation.
The reactivities of 2C5 monoclonal antibody indicate that the 16-cell mouse
embryo consists of a composite of cells of differing cell surface phenotype. The
blastomeres of differing phenotype show distinct properties on aggregation to
16-cell embryos and differ in the ability to relocate to internal and external
positions in the early blastocyst. The cosegregation of monoclonal antibody
reactivity and cell behaviour on aggregation is related to the tendency of cells to
Monoclonal antibody reactivity to early mouse embryos
277
be retained in differing relative positions and as a consequence, to contribute
to the inner cell mass and trophectoderm of the blastocyst. It is clear that cell
phenotype and cell behaviour do not commit cells to trophectoderm and inner
cell mass lineages at the 16-cell stage as indicated by experiments of Rossant &
Vijh (1980). However, the differing cell surface characters and behaviour do
mark the onset of the divergence of blastomeres to the lineages of the tissues of
the blastocyst.
I thank Chris Graham, Steve Gaunt, Colin Stewart and Martin Johnson for their helpful
discussions. I also thank the Smith Kline Foundation for an emergency grant to support
preparation of monoclonal antibodies to mouse teratocarcinoma and the Cancer Research
Campaign for funds to complete this study. I finally thank the Medical Research Council for
support by a Research Studentship.
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{Received 25 January 1982, revised 16 March 1982)