Download PDF

J. Embryol. exp. Morph. 90, 335-361 (1985)
335
Printed in Great Britain © The Company of Biologists Limited 1985
Saccharide structures of the mouse embryo during the
first eight days of development
Inferences from immunocytochemical studies using monoclonal
antibodies in conjunction with glycosidases
J. E. PENNINGTON,
Applied Immunochemistry Research Group, Clinical Research Centre, Watford
Road, Harrow, Middlesex, HA1 3UJ, U.K.
S. RASTAN,
Comparative Medicine Division, Clinical Research Centre, Watford Road, Harrow,
Middlesex, HA1 3UJ, U.K.
D.ROELCKE
Institute for Immunology and Serology, University of Heidelberg, Heidelberg 1,
Federal Republic of Germany
ANDT.
FEIZI
Applied Immunochemistry Research Group, Clinical Research Centre, Watford
Road, Harrow, Middlesex, HA1 3UJ, U.K.
SUMMARY
Monoclonal anti-carbohydrate antibodies have been used in conjunction with glycosidases in
immunofluorescence studies to derive information about the structures and in situ distribution of
saccharides of the mouse embryo during the first 8 days of development. The salient findings are
as follows:
(a) Branched poly-N-acetyllactosamine sequences of L-antigen type are detectable from the
first day onwards and are widely distributed in cells of the endoderm, ectoderm and mesoderm.
(b) Linear poly-N-acetyllactosamine sequences of i-antigen type are detectable from the fifth
day onwards in cells of all three lineages, but have a more restricted distribution than the
sequences of I-type.
(c) Poly-N-acetyllactosamine sequences that are susceptible to digestion with endo-/3galactosidase are the main carriers of the SSEA-1, C14 and the blood group B-like antigens,
which have the following structures
Galj81-4GlcNAc,
I 1,3
Fucor
Gal/31-4GlcNAc and Galarl-3Gal,
I 1,2
I 1,3
Fucor Fuca
respectively
and are found in endoderm and ectoderm but not in mesoderm cells. In the trophoblast
however, these antigens are borne on saccharides that are resistant to endo-j8-galactosidase.
Key words: carbohydrate structures, carbohydrate antigens, embryonic antigens, differentiation
antigens, mouse embryo, saccharides, immunocytochemistry, monoclonal antibodies,
glycosidases.
336
J. E . P E N N I N G T O N , S. R A S T A N , D . R O E L C K E AND T.
FEIZI
(d) A proportion of the poly-N-acetyllactosamine structures in the endoderm and the
ectoderm of the 5- and 6-day embryos may contain the following novel structures:
Galarl-Gal/31-4GlcNAc and Galorl-Galj81-4GlcNAc
I 1,3
| 1,2
| 1,3
Fucar
Fuca Fucar
in which antigenicities of SSEA-1 and C14 determinants are masked.
(e) There are several types of sialyl-oligosaccharides: (1) those reactive with anti-Gd, which
has a specificity for NeuAccv2-3Galj31-4GlcNAc sequence in the extraembryonic mesoderm
and the heart; (2) those reactive with anti-Pr2 but not with anti-Gd, which may correspond to
other N-acetylneuraminic acid containing sequences such as NeuAcar2-3Gal/31-3GalNAc or
NeuAca2-6Gal in preimplantation embryos and in the yolk sac, neural ectoderm and
mesenchyme of the 8-day embryo; (3) those with other sialic acid forms or linkages that do not
react with anti-Gd and Pr2; among these are sialosyl-i sequences in the extraembryonic
ectoderm, and sialosyl-I sequences in most cell types during the first 8 days. The latter are the
main poly-N-acetyllactosamine structures in the neural ectoderm of the 8-day embryo.
(f) The sequence Galj31-3GlcNAc/31-3GaljSl-4Glc/GlcNAc, or cross-reactive structures,
which bind FC10.2 antibody occur in the extraembryonic endoderm and yolk sac.
The roles of specific carbohydrate structures as receptors during embryonic development and
cell growth are important topics of current research. The cytochemical approach with
monoclonal antibodies in conjunction with glycosidases, as in the present study, provides a
unique opportunity to visualize the in situ disposition of specific carbohydrate sequences in
individual cells of the whole organism, and should facilitate systematic investigations of their
functions.
INTRODUCTION
Early mouse embryos and embryonal carcinoma cells are rich in glycans with
poly-N-acetyllactosamine (Gal/31-4GlcNAc/31-3)n sequences (Muramatsu et al.
1978). Immunocytochemical studies with the natural monoclonal antibodies, anti-I
and anti-i have shown that in the developing mouse embryo and in embryonal
carcinoma cells which are induced to differentiate in vitro, stage-specific changes
occur in the branching patterns of these saccharides (Kapadia, Feizi & Evans,
1981; Feizi, Kapadia, Gooi & Evans, 1982). Branched type 2 backbone structures
(I-antigen type) are detectable at the zygote stage and throughout the first 6 days
of gestation, while linear structures of i-antigen type are first detectable at the
onset of differentiation of primary endoderm cells. The hybridoma-defined stagespecific embryonic antigen, SSEA-1 (Solter & Knowles, 1978) consists of 3fucosyl-N-acetyllactosamine (Gooi et al. 1981; Hounsell, Gooi & Feizi, 1981). All
three antigens (I, i and SSEA-1) are masked in the presence of the blood group Hassociated arl-2 linked fucose (Feizi et al. 1971; Gooi et al. 1981; Gooi et al. 1983b)
or in the presence of sialic acid (Gooi et al. 1983b). Thus some of the sequential
changes in antigenicity that occur during embryogenesis may result from changes
in glycosylation of glycoproteins and. glycolipids (Feizi, 1981a), analogous to the
developmentally regulated changes that occur on human erythrocytes (Feizi,
1981b; Hakomori, 1981). Several other hybridoma-defined saccharide antigens of
the blood group series change during the development of mouse embryos
(Blaineau et al. 1983) or the differentiation of human embryonal carcinoma cells
(Gooi et al. 1983d), as do the major blood group antigens A, B and H in various
Saccharide structures of the mouse embryo
2>2>1
organs of the human foetus (Szulman, 1980), and the B-like structures reactive
with B. simplicifolia lectin in the mouse embryo (Wu, Wan & Damjanov, 1983).
However, such developmental changes are not confined to the blood group family.
Studies with monoclonal antibodies have shown that during mouse embryonic
development, changes also occur on carbohydrate chains of glycolipids of the
globo series which express the Forssman (Stern et al. 1978; Willison et al. 1982),
SSEA-3 and SSEA-4 (Kannagi et al. 1983a,b) antigens. Thus, monoclonal antibodies have been invaluable in providing structural information on the surface
carbohydrates of embryonic cells which cannot be readily characterized structurally on account of their limited amounts and marked heterogeneity.
In view of the association of several of the carbohydrate differentiation antigens
with receptor systems (reviewed by Feizi & Childs, 1985), it is of considerable
interest to visualize their in situ disposition in individual cells and tissues during
embryogenesis. In the present studies several hybridoma antibodies against blood
group related oligosaccharides have been used in conjunction with monoclonal
human autoantibodies anti-I, i and glycosidases to derive new information on the
backbone and peripheral regions of saccharides of the cells of pre- and postimplantation mouse embryos up to the eighth day of development. In addition,
two human monoclonal autoantibodies recognizing N-acetylneuraminic-acid-containing structures have provided information on changes in sialylation during
development.
MATERIALS AND METHODS
Antibodies
Four monoclonal antibodies with anti-I or I-like specificities were used (Table 1). Anti-I Ma,
anti-I Step and anti-i Den are human antibodies (Feizi, 198LZ?) and the anti-I-like antibodies M39
and M18 (Gooi et al. 1983c) are mouse hybridoma antibodies [Foster, Edwards, Dinsdale &
Neville (1982) gifts of Dr P. A. W. Edwards, Ludwig Institute for Cancer Research, Sutton,
U.K.]. IgM w o ° (gift of Dr E. Osserman, Columbia Medical Center, New York), is a
Waldenstrom macroglobulin with specificity for the type 1 (Gal/81-3GlcNAc)-based backbone
sequence (Kabat, Liao, Shyong & Osserman, 1982). Anti-Pr2 (designated LTh) and anti-Gd
(designated Kn) are human monoclonal antibodies which recognize the NeuAc-Gal sequence
(Table 1). The former reacts with both the <*2-3 and ar2-6 linked sequence whereas the latter
reacts only with the a2-3 linked sequence (Uemura, Roelcke, Nagai & Feizi, 1984). The
specificities of the mouse hybridoma antibodies anti-SSEA-l (Gooi et al. 1981; Hounsell et al.
1981, gift of Dr D. Solter, The Wistar Institute, Pennsylvannia, U.S.A.), Hll (Knowles, Bai,
Daniels & Watkins, 1982, gift of Dr W. M. Watkins, Clinical Research Centre, Harrow, U.K.),
TL5 (Gooi et al. 1983a, gift of Dr J. Schlessinger, Weizmann Institute, Rehovot, Israel), C14
(Brown et al. 1983, gift of Professor R. W. Baldwin, Nottingham University, U.K.) and FC10.2
(Gooi et al. 1983d, gift of Dr R. A. J. Mcllhinney, Ludwig Institute, Sutton, U.K.) for the
respective structures, 3,fucoslyl-N-acetyllactosamine (Lex antigen), the blood group H
structure, the blood group A structure, 2'3-difucosyl-N-ac:etyllactosamine (Ley antigen) and the
type 1 based backbone sequence are shown in Table 1. The monoclonal anti-B antibody
(NB10/3B4, gift of Dr E. Lennox, Laboratory of Molecular Biology, Cambridge, U.K.) has
been cited by Voak et al. 1983. This antibody reacts (H. C. Gooi, unpublished observations)
equally well with untreated and defucosylated group B ovarian cyst substances [mild acidtreated under conditions which remove fucose residues (Gooi etal. 1983b)]. Thus we deduce that
the specificity involves the B-like sequence shown in Table 1. The isolectin BS I-B4 from
Bandeiraea simplicifolia, conjugated with fluorescein isothiocyanate was purchased frdm Sigma
338
J. E. PENNINGTON, S. RASTAN, D. ROELCKE AND T. F E I Z I
Table 1. Carbohydrate sequences known to react with the antibodies and lectin used in
the present studies
Carbohydrate structures known to react
Antibodies/Lectin
Anti-I
Gal/51-4GlcNAc)8K.
s
JjGal/Sl-4GlcNAc/SlMa
M18
M39
Step
Ma
M18
M39
Gal)81-4GlcNAc/Sls
6
Gal/fr-4GlcNAc/Sl N A
~ N v,Gal/31-4GlcNAc/31" " - ^ N ^Gal/n-4GlcN Ac/ft ^
Gal/31-4GlcNAc/Sl
6Gal/Man (GalNAc)
Anti-i Den
Gal)81-4GlcNAcj81-3Gal/Sl-4GlcNAc/Sl-3Gal/31-4GlcNAc/3l-
Anti-SSEA-1
(anti-'LeX))
Gal/Sl-4GlcNAcj8Fucar
H l l (anti-H)
Gal)81-4GlcNAcj8-
11,2
Fucar
C14 (anti-'Ley>)
Gal/31-4GlcNAc/JFucar
Fucar
wo
IgM °
FC10.2
Gal)81-3GlcNAc/31-3Gal/51-4Glc/GlcNAc)8l-
Anti-Pr2
NeuAca2-3/6Gal... including brain gangliosides such as:
NeuAco2-3Gal/31-4Glc-Cer
Gal/31-3GalNAc/31-4Galj81-4Glc-Cer
|2,3
NeuAcar
(GM1)
NeuAca2-3Gal/Sl-3GalNAcj81-4Gal/Sl-4Glc-Cer
|2,3
NeuAco"
(G Dla )
Anti-Gd
NeuAca2-3Gal/31-4GlcNAc/31-(3Gal/Sl-4GlcNAc/Sl-)n
TL5 (anti-A)
GalNAcarl-3Galj81-3 /4GlcNAc/31
|
| | /
± Fucar ± Fucar
NB10/3B4
(anti-B)
Galal-3Gal/3|1,2
(± Fuca)
BS I-B4 lectin
Galarl-3Gal/S-
The determinants on the branched poly-N-acetyllactosamine sequences recognized by anti-I Ma, M18 and
M39 antibodies are indicated by dotted underlining and those recognized by anti-I Step, solid underlining.
Anti-I Ma and Step and anti-i Den can react, whereas M18 and M39 cannot react, with their antigenic
determinants in the presence of peripheral galactose residues joined by arl-3 linkage to the terminal /Sl-4
linked galactoses. Reactivities with the anti-I, anti-i, M18 and M39 antibodies are masked in the presence of
terminal sialic acid residues. Anti-Pr2 shows a preferential reaction with the short chain glycolipid G M3
although it can also react with the poly-N-acetyllactosamine based structures; in addition this antibody reacts
with sialoglycolipids of the ganglio series which do not react with anti-Gd; this latter antibody has a
preferential reaction with a2-3 sialylated poly-N-acetyllactosamine sequences. Anti-Pr2 and anti-Gd do not
react with N-glycolylneuramic acid-containing sequences.
Saccharide structures of the mouse embryo
339
Chemical Company (Poole, Dorset, U.K.). This lectin also recognizes the blood group B-like
sequence (Wood, Kabat, Murphy & Goldstein, 1979).
The human antibodies were used as plasma dilutions: anti-I Ma (1:100) anti-I Step (1:700)
anti-i Den (1:700) anti-Pr2 (1:50), anti-Gd (1:10). Mouse hybridoma ascites containing antiSSEA-1, H l l , M18 and M39 antibodies were used at 1:100 dilution. Antibody TL5 was used as
an IgG preparation at 6 fig ml"1. Antibodies C14, NB10/3B4 and FC10.2 were used as undiluted
culture supernatants. The isolectin BS I-B4 was used for immunofluorescence at 200jugml~1;
lectin in the presence of 0-lM-D-galactose was used as a negative control. Normal human
serum, supplemented with human IgM (Feizi, Kapadia & Yount, 1980), mouse ascites
containing an irrelevant monoclonal antibody (anti-house dust-mite) and Dulbeccos MEM
medium with 10 % foetal calf serum were also used as appropriate negative controls.
Embryos
6- to 8-week-old (C57Bl/10xCBA)F! female mice were superovulated with 5i.u. pregnant
mare serum gonadotrophin (Intervet, Science Park, Middleton, Cambridge, U.K.), followed
44h later by 5i.u. human chorionic gonadotrophin (HCG, Intervet). They were caged with
(C57Bl/l0xCBA)F1 male mice and checked the following morning for the presence of
copulation plugs to confirm mating. Ovulation was assumed to occur 12 h post HCG. The day of
copulation plug was designated day 1 of pregnancy.
Preimplantation embryos were flushed from the oviducts 24 and 48 h post HCG at the zygote
and 2- to 4-cell stages respectively, using a Hepes-buffered medium M2 (Quinn, Barros &
Whittingham, 1982) containing 4 mg bovine serum albumin (BSA) ml"1. Embryos were cultured
at 37°C until the morula stage in microdrops of medium M16 (Whittingham, 1971) containing
4mg BSA ml"1 and overlaid with paraffin oil in Falcon tissue culture dishes in a humidified
atmosphere of 5 % CO2 in air. Manipulations of embryos were carried out in Hepes-buffered
M2 under a Wild dissecting microscope. Zygotes were freed from cumulus cells by 1-2 min
incubation with a lOOmgrnl"1 solution of hyaluronidase (Sigma Chemical Company, Poole,
U.K.) in 001 M-phosphate-buffered saline pH7-4. The zonae pellucidae were removed by
treating embryos for 5 min at 37°C with a 5 ing ml"1 solution of Pronase (Calbiochem-Behring
Corp., La Jolla, California, U.S.A.) in 0-01 M-phosphate-buffered saline pH7-4 containing
10mg polyvinyl pyrrolidinemP1 (Calbiochem-Behring Corp.), for 5min at 37°C, followed by
three washes in M2 medium. Previous time-course experiments (Rastan et al. 1985) show that
the expression of the I and SSEA-1 antigens is unaffected by treatment of the embryos with
Pronase for 5-15 min. In exploratory experiments we observed a similar antigenicity of the
embryos whether they were tested in suspension or as sections after fixation; moreover the
antigenicity of cultured 1-cell to 16-cell embryos was the same as that of embryos tested directly
after flushing from the oviducts.
Postimplantation embryos in their decidua were dissected from the oviduct at 5 to 8 days
gestation, fixed in 10% v/v neutral formalin, embedded in paraffin and serially sectioned
at 4^m thickness. Sections were dewaxed in xylene and hydrated through absolute to 70%
v/v alcohol and washed in phosphate-buffered saline before enzyme treatment and immunofluorescence staining.
Glycosidases
Endo-/S-galactosidase (gift of Dr P. Scudder) was isolated from culture filtrates of Bacteroides
fragilis (Scudder et al. 1983; Scudder, Hanfland, Uemura & Feizi, 1984). Knowledge on the
specificity of this enzyme, namely its ability to cleave internal /3-galactosyl linkages in
unbranched domains of poly-N-acetyllactosamine sequences but not those adjacent to branch
points has been summarized by Rastan et al. (1985). Treatment of preimplantation embryos with
this enzyme, and the effects on antigenicity have been described elsewhere (Rastan et al. 1985)
and the results are cited in this report for completeness. Sections of postimplantation embryos
were incubated with this enzyme (2i.u. ml"1) dissolved in 50mM-sodium acetate pH5-8
containing O^mgml" 1 BSA (incubation conditions are given below). For removal of terminal
sialialic acid residues, sialidase from Vibrio cholerae (Behringwerke, AG, Marburg, W.
Germany) dissolved in 50mM-sodium acetate pH5-5, at li.u.ml" 1 was used for postimplantation embryos and from Clostridium perfringens (Sigma) reconstituted in M2 medium at
340
J. E . P E N N I N G T O N , S. R A S T A N , D . R O E L C K E AND T.
FEIZI
2i.u. ml" 1 for preimplantation embryos. Coffee bean a-galactosidase (Sigma) suspended in
ammonium sulphate lOi.u. ml"1 was diluted in 50mM-phosphate buffer pH6-5, and used at
1 i.u. ml"1 on postimplantation embryos. The preimplantation embryos were incubated with this
enzyme (2i.u. ml"1) which had been dialysed against M2 medium for 5 h at 4°C; the activity of
this enzyme was determined before use according to the method of Li & Li (1972) using
p-nitrophenyl ar-D-galactoside as substrate.
Preimplantation embryos were incubated with enzymes for l h at 37°C in an atmosphere of
5 % CO2 in air, fixed with 4 % formaldehyde (w/v) in phosphate-buffered saline containing
0-lM-CalCl2 for l h at room temperature, and washed three times in M2 medium before
immunofluorescence. Sections of postimplantation embryos (dewaxed) were incubated for 16 h
with enzymes or control buffers at 37 °C and washed for 20min in phosphate-buffered saline at
4°C. Immunofluorescence of pre- and postimplantation embryos was performed as described by
Rastan et al. (1985) and Kapadia et al. (1981) respectively.
Individual experiments were repeated on at least two occasions.
RESULTS
Reactivities with anti-I Ma, anti-I Step and with antibody M39 and M18
Embryos from the zygote stage onwards showed intense immunofluorescence
with the anti-I antibodies Ma and Step throughout the first 7 days of
embryogenesis (Figs 1A, 6). However, reactivities with M39 and M18 antibodies
were restricted to the trophoblast and the luminal aspects of cells lining the
proamniotic cavity until the 6-day stage (Figs IB, 3,5) when the visceral endoderm
reacted weakly. On the 7th day both the embryonic and extraembryonic endoderm, the cells lining the exocoelom, and, to a lesser extent, the embryonic
ectoderm were immunostained by these two antibodies (Figs IB, 7). On the 8th
day the immunofluorescence with M39 and M18 was similar to that seen with the
anti-I antibodies (Figs IB, 7) being predominantly in cells lining various lumens,
including the yolk sac endoderm, the amnion, dorsal aorta, coelom, neural groove
and gut.
The lack of reactivity of all or part of the embryo with M39 and M18 antibodies
during the first 6 days suggested that certain peripheral glycosylations might
be hindering reactivities of the branched I-type oligosaccharides with these
antibodies. This was confirmed by immunofluorescence after treatment of the
embryos with ar-galactosidase or sialidase. With the exception of the zygote stage,
which was unaffected by ar-galactosidase treatment (Fig. 3), the preimplantation
and the 5- to 7-day embryos showed moderate or strong immunofluorescence after
treatment with either ar-galactosidase or sialidase (Figs 3, 5, 7), and the
distribution of immunofluorescence was similar to that observed with anti-I
antibodies using untreated embryos (Fig. 1A,B). At 8 days, sialidase, but not argalactosidase, revealed new immunofluorescence with M18 antibody as with anti-I
Ma and Step (see below). The giant cells of the trophoblast stained by anti-I Ma
and Step (Fig. 7) were rendered reactive with M18 antibody after sialidase
treatment (Fig. IB).
Neither enzyme treatment affected the immunofluorescence with anti-I Ma and
Step during the first 7 days of embryogenesis (immunofluorescence of the
untreated cells was already so intense that an increase of immunoreactivity might
Saccharide structures of the mouse embryo
341
not be detectable). However, treatment in the 8-day embryo with sialidase
revealed a strong immunofluorescence of the mesenchyme, mesoderm cells of the
yolk sac, epimyocardium and of cells of the neural ectoderm at the posterior end of
the neural tube (Fig. 7); treatment with ar-galactosidase was without effect.
The relative intensities of immunofluorescence with M18 revealed by the two
glycosidases and the increased reactivities with anti-I antibodies after sialidase
treatment of the 8-day embryo are consistent with the presence of a higher
proportion of sialylated branched poly-N-acetyllactosamine structures during the
1st and 2nd days and the 7th and 8th days of development. This contrasts with a
higher proportion of a'-galactosyl termini during the 3rd to the 6th days of
development.
Reactivities with anti-i antibody Den
Immunofluorescence with this antibody was first detectable in the parietal and
visceral endoderm of the 5-day embryo (Fig. 1A) and was not affected by
pretreatment with <*-galactosidase or sialidase. However at 6 days, the reactivity
of the extraembryonic ectoderm with anti-i Den was substantially enhanced by
sialidase treatment (Fig. 5), consistent with the presence of sialosyl-i sequences. In
the 7-day embryo, both layers of the amnion and the cells lining the amniotic
cavity and the exocoelom (Fig. 6) and cells in the mesoderm layer of the chorion
were strongly stained, whereas those lining the ectoplacental cavity were not.
Primary giant cells of the trophectoderm also reacted with this antibody at this
stage. This immunofluorescence was not affected by pretreatment with either agalactosidase or sialidase.
On the 8th day, reactivities with anti-i Den were similar to those with the anti-I
antibodies except that secondary giant cells did not react with this antibody, and
no additional reactivities were revealed either after sialidase or ar-galactosidase
treatment (Fig. 1A).
Reactivities with anti-SSEA-1 and C14 antibodies
In contrast to the extensive immunofluorescence with anti-SSEA-1, only two
out of a dozen 8-cell embryos and only small areas of the 5-day embryo showed
immunofluorescence with C14 antibody (Figs 2A,B, 4). However at the 6-day
stage, the distribution of immunofluorescence with C14 antibody resembled that
seen with anti-SSEA-1. On days 7 and 8, reactivity with C14 antibody was again
more restricted (Fig. 6). For example, cells of the embryonic ectoderm on day 7
(Fig. 6 insets) or neural ectoderm (on day 8) reacted only with anti-SSEA-1 (Fig.
2A,B).
Pretreatment of the embryo sections with sialidase did not affect immunofluorescence with these two antibodies (results not shown). However, pretreatment with ar-galactosidase resulted in a substantial increase in immunofluorescence of the 8-cell embryo (Fig. 3) and the embryonic ectoderm of the
5- and 6-day embryos with anti-SSEA-1 and the endoderm of the 5- and 6-day
Ii
Ii
Ii
Embryonic
endoderm
Gut
Ii
Parietal
Neural
ectoderm
Embryonic
ectoderm J
Embryonic
ectoderm
Primitive
ectoderm I
[I] s
Mesenchyme
Embryonic
mesoderm
(D s
Heart
Ii
Somatopleura
Ii
I
Yolk sac
Ii
Chorion
Extraembryonic
mesoderm
Amnion-ect
Amnion-mes ^Allantois
Ii
Ectoplacental
cavity j
Extraembryonic
ectoderm
Secondary giant
ceils
Ectoplacental
cone |
Primary giant
cells |
Primary giant
cells |
Mural
Trophectoderm
DAY
1
2
3
(4)
Fig 1 The distribution of carbohydrate antigens recognized by monoclonal antibodies during the first 8 days of development of the
mouse embryo (blastocyst stage, day 4, not tested). Observations with anti-I Ma, Step and anti-i Den are shown in panel A; M39
and/or M18 in panel B; anti-Pr2 and anti-Gd in panel C. Abbreviations: I, denotes reactivity with anti-I Ma and anti-IStep; i, with
anti-i Den- M, with M39 and/or M18; P, with anti-Pr2; G, with anti-Gd; m, immunofluorescence detected with M18 or M39
antibodies only after treatment of embryos with siaUdase or *-galactosidase (i.e. cryptic determinants); [I], immunofluorescence
detected with anti-I antibodies only after treatment of embryos with siaUdase (i.e. cryptic I determinants). When immunoreactivity
was revealed or enhanced after treatment of embryos with siaUdase and ar-galactosidase, the superscripts s and g are used
respectively; curved brackets indicate weak immunofluorescence.
Yolk sac
Ii
Embryonic
visceral
endoderm
Parietal Ii
Visceral Ii
Extraembryonic
visceral
endoderm
Parietal I i
Visceral Ii
Primitive
'endoderm'
• Inner cell mass-
-Blastocyst—
8 cells I
2-4 cells I
Zygote I
C/5
W
4
to
M
Yolk sac
M
Extraembryonic Embryonic
IM visceral
visceral
• endoderm
endoderm
B
M
Embryonic
endoderm
Gut
Parietal
l\/f(s)g
• Inner cell mass
Neural
ectoderm
m
s
Embryonic
ectoderm
ms
Mesenchyme
m
M
Somatopleura
ms
Yolk sac
Extraembryonic
mesoderm
Amnion-ect ^ /
\
Amnion-mes^Allantois Chorion
(s)g
M
Blastocyst
Fig. IB
Ms
Heart
,s(g)
s(g)
8 cells m
2-4 cells
Zygote III
M
Ectoplacental
cavity
Extraembryonic
ectoderm
M
ms
Secondary giant
cells
M
Ectoplacental
cone
M
Ectoplacental
M
Primary giant y
cells
cells
M
Primary giant 6
cells
M Mural
•Trophectoderm
2
3
(4)
DAY
1
1
I
P
Yolk sac
Embryonic
visceral
endoderm
Embryonic
endoderm
Gut
Parietal
Parietal
Visceral
Extraembryonic
visceral
endoderm
Parietal
Visceral
Primitive
'endoderm'
• Inner cell mass
Neural
ectoderm
P
Mesenchyme
GP
Heart
Fig. 1C
Somatopleura
P
Yolk sac
\
Chorion
Extraembryonic
mesoderm
Ectoplacental
cavity
Extraembryonic
ectoderm
Secondary giant
cells
Ectoplacental
cone
Ectoplacental
cone
Primary giant
cells
Primary giant
cells
Mural
•Trophectoderm
2
3
(4)
>
z
o
H
GO
z
H
O
z
z
o
DAY
w
1
z
W
Saccharide structures of the mouse embryo
345
embryos with both antibodies (Fig. 4). There was no immunofluorescence of
mesoderm-derived cells with either antibody.
These observations indicate that there are terminal ar-galactosyl residues in
close proximity to the Lex antigen sequence in the 8-cell embryo and to both the
Lex and Ley antigen sequences in the 5- and 6-day embryos.
Reactivity with monoclonal anti-B antibody and Bandeiraea simplicifolia lectin
There was no immunofluorescence of preimplantation embryos with the anti-B
monoclonal antibody, NB10/3B4 (preimplantation stages were not tested with
the lectin). In postimplantation embryos there was a similar distribution of
immunofluorescence with the anti-B antibody and lectin (Fig. 2C), but the
reactions with the lectin were always weaker. The visceral endoderm was strongly
stained at the 5- and 6-day stages, and there was also weak staining of the parietal
endoderm and trophoblast at the 6-day stage (Fig. 4). On day 7, immunofluorescence was restricted to the extraembryonic endoderm and primary giant
cells of the trophoblast (Fig. 6). At the 8-day stage, reactivity with the anti-B
antibody was restricted to the primitive gut and primary giant cells of the
trophoblast (Fig. 2C). The immunofluorescence data, summarized in Figs IB and
2C, indicate that only a proportion of ar-galactosyl termini are available for
reaction with the anti-B antibody and lectin.
Effect of endo-f$-galactosidase
Additional evidence for the presence of linear poly-N-acetyllactosamine
sequences was sought by comparing the immunofluorescence with anti-I, anti-i,
M18, M39, anti-SSEA-1, C14 and anti-B antibodies before and after treatment
with endo-/3-galactosidase. In accordance with the known susceptibility of linear
poly-N-acetyllactosamine domains to digestion by endo-/3-galactosidase (Scudder
et al. 1984), immunofluorescence with anti-i Den at the 6- and 8-day stages tested
was abolished after treatment with this enzyme (results not shown). Immunofluorescence of preimplantation embryos with both anti-I antibodies Ma and Step,
was reduced but not abolished by treatment with this enzyme [these observations
are described elsewhere (Rastan etal. 1985)]. With the postimplantation embryos,
immunofluorescence with anti-I Step was slightly reduced but with anti-I Ma there
was no effect. These observations suggest that in preimplantation embryos, there
is a higher proportion of linear segments on the branched, I-active, poly-Nacetyllactosamine chains than in postimplantation embryos.
Immunofluorescence with M39 and M18 antibodies was enhanced after
treatment of the 6-day embryo with endo-jS-galactosidase (Fig. 5 inset), suggesting
that digestion of long-chain oligosaccharides of poly-N-acetyllactosamine type
resulted in the exposure of short unsubstituted Gal/31-4GlcNAc sequences which
were formerly inaccessible.
Pretreatment of both the preimplantation (Rastan et al 1985) and postimplantation embryos (Fig. 4) with endo-/J-galactosidase abolished reaction with
3
Neural
ectoderm
Embryonic
endoderm
Gut
s
s
Mesenchyme
Heart
8 cells
S
Somatopleura
Yolk sac
Chorion
Extraembryonic
mesoderm
Amnion-ect ^^AllQntm
Allantois
Amnion-mes
s
-Blastocyst
g
Ectoplacental
cavity
Extraembryonic
ectoderm
Secondary giant
cells
Ectoplacental
cone
Ectoplacental
Primary giant
« cells
Primary giant
cells
S Mural
•Trophectoderm
DAY
1
2
3
(4)
Fig. 2. The distribution of carbohydrate antigens recognized by monoclonal antibodies during the first 8 days of development of the
mouse embryo (blastocyst stage, day 4, not tested). Observations with anti-SSEA-1 and C14 are shown in panels A and B,
respectively and those with the anti-B antibody NB10/3B4 and Bandeiraea simplidfolia lectin in panel C. Abbreviations: S, denotes
reactivity with anti-SSEA-1; C, with C14 antibody, B a with the B antibody (NB10/3B4) and B L with the Bandeiraea simplidfolia
lectin.
s
Yolk sac
Cg Primitive
ectoderm
a
Embryonic
o ectoderm
Parietal
^
Parietal
Parietal
Extraembryonic Embryonic
o visceral
visceral §
endoderm
endoderm
S g Visceral
S g Visceral
Primitive
''endoderm'
. Inner cell mass-
2-4 cells
Zygote
w
H
D
5S
w
w
c
Yolk sac
c
Embryonic
endoderm
Gut
Parietal
QS Parietal
Visceral
\
Extraembryonic Embryonic
p visceral
visceral |
endoderm
endoderm
Parietal
Primitive
'endoderm'
Primitive
£ ectoderm
. Inner cell mass-
Qfi Visceral
B
Mesenchyme
Fig. 2B
Heart Somatopleura
-Blastocyst-
8 cells
2-4 cells
Zygote
Yolk sac
Chorion
Extraembrvonic
Ectoplacental
cavity
Extraembryonic
ectoderm
Secondary giant
cells
Ectoplacental
cone
Primary giant
cells
Primary giant
cells
Q Mural
•Trophectoderm
£r*
(4) i
3
DAY o,
Visceral
B
Yolk sac
Extraembryonic
visceral
endoderm
Embryonic
visceral
endoderm
B a B L Visceral
BaBL
Mesenchyme
Somatopleura
Fig. 2C
Heart
Yolk sac
Chorion
\
Extraembryonic
mesoderm
cavity
Ectoplacental
Extraembryonic
ectoderm
Primary giant
D cells
BaBL
cells
Primary giant
£>n£>i
D
Secondary giant
cells
Ectoplacental
Ectoplacental
cone
Mural
•Trophectoderm
6
5
DAY
1
2
3
(4)
-p>-
N
w
H
CO
z
o
H
o
w
Z
m
00
Saccharide structures of the mouse embryo
349
G+
GS-
S+
B
M18
SSEA-1
*'•*-.,
K
Fig. 3. lmmunofluorescence of zygote (A-C), 2-cell stage (D-F) and 8- to 16-cell
stage (G-L) embryos with M18 antibody or with anti-SSEA-1. Results with untreated
embryos are shown under G S - , those with ar-galactosidase or sialidase-treated
embryos are under columns G+ or S+ respectively. Inset in panel E shows an
additional 2-cell-stage embryo with an accentuation of immunofluorescence at sites of
contact between blastomeres. Magnification A-C x530, D-F x490, G-L x460. Inset
EX240.
350
J. E. PENNINGTON, S. RASTAN, D. ROELCKE AND T. FEIZI
anti-SSEA-1, with the exception of the trophoblast cells whose reactions were
unaffected. These observations suggest that the 3-fucosyl-N-acetyllactosamine
sequence is carried on two types of carbohydrate backbones: those with linear
03
•o
10
•
C14
o
'«
,
>'•
)
SS
•o
E-
E+
i
Fig. 4. Immunofluorescence of 5- and 6-day embryos with anti-SSEA-1 (SS), C14
antibody, or anti-B antibody, NB10/3B4 (Ba). Transverse sections were tested
untreated ( E - or G - ) or after treatment with endo-j8-galactosidase (E+) or agalactosidase (G+). Abbreviations: ee, embryonic ectoderm; pc, proamniotic cavity;
pe, parietal endoderm; t, trophoblast; ve, visceral endoderm. Magnification of 5-day
embryos X320 and 6-day embryos x270.
Saccharide structures of the mouse embryo
M18
Den
Fig. 5. Immunofluorescence of 6-day embryos with M18 antibody or anti-i Den.
Longitudinal sections were tested before treatment with ar-galactosidase or endo-/3galactosidase ( G / E - ) or sialidase (S-) and after treatment with ar-galactosidase (G+)
or sialidase (S+). Small inset E+, shows a part of the embryo stained with M18
antibody after treatment with endo-j8-galactosidase. Abbreviations: xe, extraembryonic ectoderm, other abbreviations are as in Fig. 4. x270.
351
352
J. E. PENNINGTON, S. RASTAN, D. ROELCKE AND T. FEIZI
poly-N-acetyllactosamine domains in the embryo proper and those with other,
endo-/3-galactosidase-resistant, sequences in the trophectoderm.
Similarly, the partial staining of the 5-day embryo seen with C14 antibody
(Fig. 4),,and the anti-B reactivity of 6-day (Fig. 4) and 7-day embryos (results not
shown) were markedly diminished after endo-/J-galactosidase treatment. However, immunofluorescence of primary giant cells of the trophoblast with anti-B
(tested at 7 days), was unaffected by endo-/3-galactosidase: this suggests that as
xv e
Fig. 6. Immunofluorescence of 7-day embryos. The four large panels on the left show
immunofluorescence of almost complete sections of embryos with anti-i Den (De);
anti-I Ma (Ma); anti-SSEA-1 (SS); and C14. The six panels on the right show parts of
embryo sections; the two uppermost show immunofluorescence of the extraembryonic
visceral endoderm with FC10.2 (Fc) and the anti-B antibody (Ba); the four lower
panels show the amnion reacting with anti-i Den (De), anti-SSEA-1 (SS), anti-Gd
(Gl), and the allantois with anti-Gd (G2). Insets to panels SS and C14, show
immunofluorescence of embryonic ectoderm (ee) with anti-SSEA-1, contrasting with
the lack of immunofluorescence with C14 antibody. Abbreviations: oc, amniotic cavity;
cd, allantois; am, amnion; ame, ectoderm layer of the amnion; amm, mesoderm layer of
the amnion; ch, chorion; ec, ectoplacental cavity; exo, exocoleom; pg, primary giant
cells of the trophoblast; xve, extraembryonic visceral endoderm; other abbreviations
are as in previous figures. Magnification x70 except for insets xl20 and the four small
panels lower right X130.
Saccharide structures of the mouse embryo
7 day
353
8 day
S+
Sm
ne
ye_
Fig. 7. Immunofluorescence of parts of 7 and 8 day embryos. Sections of embryos were
stained with M18 antibody or with anti-I Ma before (S-) or after (S+) treatment with
sialidase. With anti-I Ma, two areas of the 8 day embryo, cephalic (left panels) and
caudal (right panels) are shown. Immunofluorescence with anti-Pr2, anti-Gd and
FC10.2 (FC) was performed on untreated (S-) sections. Abbreviations: g, gut; h,
heart; m, mesenchyme; ne, neural ectoderm; nf, neural fold; ng, neural groove; sg,
secondary giant cells; sm, somatopleura; y, yolk sac; ye, yolk sac endoderm; ym, yolk
sac mesoderm. Other abbreviations are as in previous figures. Magnification of 7-day
embryo xl60; 8-day embryo stained with M18 x60; anti-I Ma xlOO; FC10.2 x290;
anti-Gd x!50 and anti-Pr2 xlOO.
354
J. E. PENNINGTON, S. RASTAN, D. ROELCKE AND T. FEIZI
with SSEA-1, the blood group B-like antigen is carried on more than one type of
carbohydrate chain in the developing embryo.
Reactivities with anti-Pr2 and Gd antibodies
There was patchy immunofluorescence of the preimplantation embryos with
anti-Pr2. This antibody did not react with the 5- to 7-day postimplantation
embryos, but in the 8-day embryo it reacted with the neural ectoderm, the heart
and the mesoderm cells of the yolk sac (Figs 1C, 7). The staining was abolished
after sialidase treatment (results not shown).
The reaction pattern with anti-Gd was different from that observed with antiPr 2 . Reactivity with anti-Gd was absent in the preimplantation embryos and was
first observed at the 7-day stage, on cells of mesodennal origin lining the
exocoelom and including the mesoderm-derived cells of the amnion and the
allantois (Fig. 6). At 8 days, the Gd determinant was expressed in the neural
groove and the mesoderm-derived cells of the epimyocardium (Fig. 7). These
observations suggest that there are at least two types of N-acetylneuraminic acidcontaining sequences in the mouse embryos: (a) those reactive with anti-Pr2 but
not with anti-Gd, occurring in preimplantation embryos and in several cell types in
the 8-day embryo, and (b) those preferentially reactive with anti-Gd occurring in
mesodermal cells of the 7- and 8-day embryos.
Reaction with FC10.2 antibody and lack of reaction with IgMw0°, anti-Hand anti-A
antibodies
Contrasting with the abundance of type 2 (Gal/?l-4GlcNAc)-based structures
reactive with the anti-I, anti-i, M18 and M39 antibodies, and with anti-SSEA-1,
C14 and anti-Gd, there was evidence for only a limited amount of type 1
(Gal/3l-3GlcNAc) chain. Immunofluorescence with FC10.2 was restricted to the
extraembryonic visceral endoderm of the 7-day embryo (Fig. 6) and in the yolk sac
endoderm of the 8-day embryo (Fig. 7). No additional immunofluorescence was
revealed after treatment of 6- and 8-day embryos with ar-galactosidase and
sialidase. No immunofluorescence was observed with I g M w o ° , the anti-H and the
anti-A antibodies during the first 8 days of development.
DISCUSSION
These studies provide considerable insight to the in situ distribution of specific
carbohydrate structures in embryonic tissues and the changes that take place
during the first 8 days of development. The following inferences can now be made:
Poly-N-acetyllactosamine structures persist during thefirst8 days of development
Immunofluorescence with anti-I Ma and Step and anti-i Den has shown that
poly-N-acetyllactosamine sequences are widely distributed in cells of the mouse
Saccharide structures of the mouse embryo
355
embryo throughout the first 8 days of development. As summarized in Fig. 1A,
branched poly-N-acetyllactosamine (I-type) structures occur throughout the first 8
days of development and the linear structures (i-type) are detectable from the 5th
day onward in endoderm and mesoderm-derived cells and the extraembryonic
ectoderm, but hardly at all in the embryonic ectoderm.
Poly-N-acetyllactosamine sequences are capped to varying degrees with sialic acid or alinked galactose residues during the first 8 days of development
Studies with two antibodies (anti-I and M18) before and after treatments with ocgalactosidase or sialidase, indicate that considerable changes occur in the capping
of the poly-N-acetyllactosamine sequences with sialic acid and ar-linked galactose,
during the first 8 days of development (Fig. 1A,B). The majority of these
carbohydrate backbones are capped with these residues during the first 6 days of
development, whereas, an abundance of uncapped sequences, accessible to M18
and M39 antibodies, is evident at 7 and 8 days. Furthermore, the relative
proportions of chains capped with sialic acid and. ar-galactosyl residues change
during development [deduced from the observations with M18 and M39 antibodies
(Fig. IB), anti-SSEA-1, C14 and the anti-B antibody (Figs 1C, 2B,C)]; there is a
higher proportion of sialylated chains at the first 2 days and the 7th and 8th days of
development, contrasting with a higher proportion of ar-galactosyl substitutions
during the 3rd to the 6th days. The observations of Fenderson, Hahnel & Eddy
(1983), who used two monoclonal antibodies reactive with N-acetyllactosamine,
are also consistent with the presence of sialylated N-acetyllactosamine sequences
in pre- and postimplantation embryos.
Poly-N-acetyllactosamine sequences are carriers of the SSEA-1, C14 (Lex) (Ley) and
B-like antigens
The loss of immunofluorescence with anti-SSEA-1, C14 and NB10/3B4
antibodies after treatment of embryos with endo-/3-galactosidase indicates that the
antigenic determinants they recognize are borne predominantly on backbone
structures of poly-N-acetyllactosamine type, containing linear domains susceptible
to digestion with this enzyme (Scudder et al 1984). Only in the trophoblast were
the SSEA-1 and B-like activities resistant to digestion with this enzyme, suggesting
that they are borne on highly branched poly-N-acetyllactosamine or other types of
carbohydrate backbone structures.
Evidence for three types of chains with terminal oc-linked galactose residues
There are at least three types of carbohydrate structure terminating with agalactosyl residues. Firstly, there is evidence for the presence of branched poly-Nacetyllactosamine structures reactive with the anti-i antibodies but unreactive with
M18 antibody unless the embryos are pretreated with ar-galactosidase. Secondly,
there is evidence suggestive of the presence of terminal ar-galactosyl residues
356
J. E. PENNINGTON, S. RASTAN, D. ROELCKE AND T. FEIZI
which mask the reactivities of SSEA-1 and C14 determinants. Thus, novel
structures such as the following:
Galarl-Galj31-4GlctfAc
I 1,3
Fucar
Galarl-Gal)31-4GlcNAc
| 1,2
| 1,3
Fucar Fucar
may be present during the 5th and 6th days of embryogenesis, and distributed as
indicated in Fig. 2B,C. Thirdly, in preimplantation embryos and the ectoderm of
5- and 6-day embryos there is evidence for the presence of short GalarlGalj81-4GlcNAc sequences which do not react with NB10/3B4 antibody, and on
which reactivities with M18 antibody are revealed after treatment of the embryos
with ar-galactosidase.
Evidence for several types of sialyloligosaccharide sequences
This study provides evidence for the presence of several types of sialyloligosaccharide sequences in the developing mouse embryo. The reaction patterns
of anti-Pr2 and anti-Gd (Fig. 1C) suggest that the mesodermal tissues of the 7-day
embryo, including the mesoderm layer of the amnion, the allantois and chorion
contain the NeuAcar2-3(Gal/31-4GlcNAc/31-3)n sequence in quantities that are
only detectable with anti-Gd. The Pr2 reactivities of the preimplantation embryos
and of the neural ectoderm, mesenchyme and yolk sac of the 8-day embryo may
reflect their content of sialoglycolipids of the ganglio series which have been
detected in embryonal carcinoma cells of the mouse (Coulon-Morelec & BucCaron, 1981; K. Uemura, J.P., D.R. & T.F., unpublished observations). It is
possible that the sialosyl-i sequence detected in the extraembryonic ectoderm on
day 6 (Fig. 1A) and the sialosyl-I sequences inferred to be in the endoderm and
ectodermal tissues of the embryo during days 5 and 6 (Fig. 1A), contain N-glycolyl
or other forms of sialic acid, or they may contain N-acetylneuraminic acid with
other types of linkage to the backbone structures that are not reactive with anti-Pr2
and anti-Gd.
A lack of expression of the H and A antigens during the first 8 days of development
The reactions with C14 antibody indicates that 2'3-difucosyl type 2 chains are
present in the embryo during days 5 to 8 of development. However, no immunofluorescence with the anti-H monoclonal and polyclonal antibodies was observed
suggesting that the majority of chains with Fucad-2Gal/31-4GlcNAc sequence
also contain fucose arl-3 linked to N-acetylglucosamine which hinders reactivities
with the anti-H antibody. There was also a lack of immunofluorescence with the
monoclonal anti-A antibody, (TL5), which can react with mono- and difucosyl
blood group A antigens, as well as non-fucosylated 'A-like' structures (Table 1).
Saccharide structures of the mouse embryo
357
TL5 antibody also reacts (Gooi et al. 1983a) with the terminal disaccharide
sequence of the Forssman antigen, GalNAcad-3GalNAc-, which was previously
detected, using monoclonal antibody Ml/22.25, on the surface of pre- and
postimplantation embryos (Stinnakre, Evans, Willison & Stern, 1981). The lack of
reactivity of the paraffin-embedded sections used in the present study, with
antibody TL5 may be due, in part, to the extraction of glycolipids by solvents used
in the dewaxing procedures (Limas & Lange, 1982; Thorpe & Feizi, 1983). Thus, it
will be necessary to test the reactivities of cryostat sections and suspensions of
unfixed cells, with this and other antibodies for the reliable assessment of
glycolipid-associated carbohydrate antigens.
Possible occurrence of type 1 backbone sequences
The immunofluorescence of FC10.2 antibody raises the possibility that some
type 1 backbone structures may be present in the extraembryonic visceral
endoderm and yolk sac. However, there was no reactivity with I g M w o ° which has
a specificity similar to that of FC10.2. These observations require biochemical
proof for we are unaware of previous reports of the presence of type 1 chains in
mouse tissues. An alternative possibility is that there exist other structures which
can cross react with this antibody but not with I g M w o ° .
The value and limitations of monoclonal antibodies in analysis of the glycoconjugates
of cells
The structural inferences are dependent on information available on the
reactions of each antibody with structurally characterized glycans. Thus, the
precision with which structural assignments can be made depend, on the one hand,
on the availability of the relevant oUgosaccharide or glycolipid haptens for testing
with each antibody, and, on the other, they reflect the degree of cross reactions of
different carbohydrate structures with a given monoclonal antibody. Whereas the
anti-I, anti-i and anti-SSEA-1 antibodies seem highly specific for the type 2 based
backbone structures (Feizi, 1981a,b\ Gooi et al. 1984) and their arl-3 fucosylated
derivatives (Hounsell et al. 1981), respectively, anti-Pr2 has a broader specificity
(as discussed above).
The special virtue of the immunocytochemical approach is the ability to clearly
visualize the differences in the saccharides of single cells and of various cell types
within a single tissue. For example, there are considerable differences in the
saccharide antigens of the two cell layers of the amnion and those of the chorion.
As summarized in Figs 1, 2 and 6, both layers of the amnion have branched and
linear poly-N-acetyllactosamine antigens, whereas the fucose containing antigens
are found only on the ectoderm layer, and poly-N-acetyllactosamine chains with
N-acetylneuraminic acid were detected only in the mesoderm layer of the amnion.
In the chorion, both layers have poly-N-acetyllactosamine sequences with Nacetylneuraminic acid termini, and there is evidence that both layers contain
branched poly-N-acetyllactosamine structures; however, linear poly-N-acetyllactosamine structures were detected predominantly in the mesoderm layer.
358
J. E. PENNINGTON, S. RASTAN, D. ROELCKE AND T. F E I Z I
These differences in distribution should be taken into account when studying the
functions of the various carbohydrate antigens in embryonic development.
However the immunocytochemical approach does not distinguish carbohydrate
structures synthesized by the cells with which they are associated from those on
glycoproteins and glycolipids that are absorbed or endocytosed. Biosynthetic
experiments will be required to distinguish these possibilities.
Biological roles of carbohydrate structures
Knowledge of the functions of these saccharides is still in its infancy. However,
recent observations suggest that carbohydrate structures of the poly-N-acetyllactosamine series may have roles in cell interactions during early embryogenesis
(reviewed by Rastan et al. 1985). The major carriers of these sequences in
embryonal carcinoma cells are proteins of high relative molecular mass (Childs et
al. 1983). This family of carbohydrate structures has also been detected on the
receptor for epidermal growth factor, and an antibody against one of these
saccharides has been reported to act as a growth factor agonist and another as an
antagonist (reviewed by Childs et al. 1984; Feizi & Childs, 1985). On the other
hand, the major carriers of the Pr2 determinants on embryonal carcinoma cells are
gangliosides (Uemura, J.P., D.R. & T.F., unpublished observations). There is
evidence that the gangliosides G M3 and G M1 may modulate the phosphorylation
and hence the function of the receptor for platelet-derived growth factor of a 3T3
cell line (Bremer etal. 1984). Both of these gangliosides are among those that react
with anti-Pr2, and the former also reacts weakly with anti-Gd (Uemura et al. 1984,
Table 1) and their levels are known to change in embryonal carcinoma cells which
have been induced to differentiate in vitro (Coulon-Morelec & Buc-Caron, 1981;
Uemura et al, unpublished observations). Thus the roles of specific saccharide
sequences during embryonic development and cell growth, and as determinants of
the tropisms of infective agents, are important topics of current research (Feizi,
1982, 1985; Feizi et al. 1984) and monoclonal antibodies should greatly facilitate
future studies in this field.
J.P. was supported by the Cancer Research Campaign. The authors are grateful to colleagues
who have provided monoclonal antibodies, to Drs E. F. Hounsell, H. C. Gooi and P. Scudder
for their constructive criticisms and to Sheila Brown for technical assistance.
REFERENCES
C , LE PENDU, J., ARNAUD, D., CONNAN, F. & AVNER, P. (1983). The glycosidic
antigen recognized by a novel monoclonal antibody, 75. 12, is developmentally regulated on
mouse embryonal carcinoma cells. EMBOJ. 2, 2217-2222.
BREMER, E. G., HAKOMORI, S., BOWEN-POPE, D. F., RAINES, E. & Ross, R. (1984). Gangliosidemediated modulation of cell growth, growth factor binding and receptor phosphorylation.
/. biol. Chem. 259, 6818-6825.
BROWN, A., FEIZI, T., GOOI, H. C , EMBLETON, M. J., PICARD, J. K. & BALDWIN, R. W. (1983). A
monoclonal antibody against human colonic adenoma recognizes difucosylated Type 2 blood
group chains. Biosci. Reps. 3, 163-170.
BLAINEAU,
Saccharide structures of the mouse embryo
359
CHILDS, R. A., PENNINGTON, J., UEMURA, K., SCUDDER, P., GOODFELLOW, P. N., EVANS,M. J. &
FEIZI, T. (1983). High molecular weight glycoproteins are the major carriers of the
carbohydrate differentiation antigens I, i and SSEA-1 of teratocarcinoma cells. Biochem.
J. 215, 491-503.
CHILDS, R. A., GREGORIOU, M., SCUDDER, P., THORPE, S. J., REES, A. R. & FEIZI, T. (1984). Blood
group active carbohydrate chains on the receptor for epidermal growth factor of A431 cells.
EMBOJ. 3, 2227-2233.
COULON-MORELEC, M. J. & BUC-CARON, M. H. (1981). Lipid patterns of embryonal carcinoma
cell lines and their derivations: Changes with differentiation. Devi Biol. 83, 278-290.
FEIZI, T. (1981a). Carbohydrate differentiation antigens. Trends Biochem. Sci. 6, 333-335.
FEIZI, T. (1981fc). The blood group Ii system: A carbohydrate antigen system defined by
naturally monoclonal or oligoclonal autoantibodies of man. Immunol. Commun. 10,127-156.
FEIZI, T. (1982). The antigens Ii, SSEA-1, and ABH are an interrelated system of carbohydrate
differentiation antigens expressed on glycosphingolipids and glycoproteins. In New Vistas in
Glycolipid Research. Adv. Exp. Med. & Biol. 152 (ed. A. Makita, S. Handa, T. Taketomi & Y.
Nagai), pp. 167-177. New York: Plenum Press.
FEIZI, T. (1985). Demonstration by monoclonal antibodies that carbohydrate structures of
glycoproteins and glycolipids are onco-developmental antigens. Nature 314, 53-57.
FEIZI, T., KABAT, E. A., VICARI, G., ANDERSON, B. & MARSH, W. L. (1971). Immunochemical
studies on blood groups XLIX. The I antigen complex: specificity differences among anti-I
sera revealed by quantitative precipitin studies; partial structure of the I determinant specific
for one anti-I serum. /. Immunol. 106,1578-1592.
FEIZI, T. & CHILDS, R. A. (1985). Carbohydrate structures of glycoproteins and glycolipids as
differentiation antigens and tumour associated antigens and components of receptor systems.
Trends Biochem. Sci. 10, 24-28.
FEIZI, T., CHILDS, R. A., HAKOMORI, S. & POWELL, M. E. (1978). Blood group Ii active
gangliosides of human erythrocyte membranes. Biochem. J. 173, 245-254.
FEIZI, T., Gooi, H. C., LOOMES, L. M., SUZUKI, Y., SUZUKI, T. & MATSUMOTO, M. (1984). Cryptic
I antigen activity and Mycoplasma pneumoniae receptor activity associated with sialoglycoprotein GP-2 of bovine erythrocyte membranes. Biosci. Rep. 4, 743-749.
FEIZI, T., KAPADIA, A., Gooi, H. C. & EVANS, M. J. (1982). Human monoclonal autoantibodies
detect changes in expression and polarization of the Ii antigens during cell differentiation in
early mouse embryos and teratocarcinomas. In Teratocarcinoma and Embryonic Cell
Interactions (ed. T. Muramatsu, G. Gachelin, A. A. Moscona & Y. Ikawa), pp. 201-215.
Tokyo: Japan Scientific Societies Press & Academic Press.
FEIZI, T., KAPADIA, A. & YOUNT, W. J. (1980). I and i antigens of human peripheral blood
lymphocytes cocap with receptors for concanavalin A. Proc. natn Acad. Sci., U.S.A. 77,
376-380.
FENDERSON, B. A., HAHNEL, A. C. & EDDY, E. M. (1983). Immunohistochemical localization of
two monoclonal antibody-defined carbohydrate antigens during early murine embryogenesis.
Devi Biol. 100, 318-327.
FOSTER, C. S., EDWARDS, P. A. W., DINSDALE, E. A. & NEVILLE, A. M. (1982). Monoclonal
antibodies to the human mammary gland. I. Distribution of determinants in non-neoplastic
mammary and extra mammary tissues. Virchows Arch. (Pathol. Anat.) 394, 279-293.
Gooi, H. C , FEIZI, T., KAPADIA, A., KNOWLES, B. B., SOLTER, D. & EVANS, M. J. (1981). Stage
specific embryonic antigen SSEA-1 involves arl-3 fucosylated Type 2 blood group chains.
Nature 292,156-158.
Gooi, H. C , SCHLESSINGER, J., LAX, I., YARDEN, Y., LIBERMANN, T. A. & FEIZI, T. (1983a).
Monoclonal antibody reactive with the human epidermal-growth-factor receptor recognizes
the blood-group-A antigen. Biosci. Rep. 3, 1045-1052.
Gooi, H. C., THORPE, S. J., HOUNSELL, E. F., RUMPOLD, H., KRAFT, D., FORSTER, O. & FEIZI, T.
(1983/?). Marker of peripheral blood granulocytes and monocytes of man recognized by two
monoclonal antibodies VEP8 and VEP9 involves the trisaccharide 3-fucosyl-Nacetyllactosamine. Eur. J. Immunol. 13, 306-312.
Gooi, H. C , UEMURA, K., EDWARDS, P. A. W., FOSTER, C. S., PICKERING, N. & FEIZI, T. (1983C).
Two mouse hybridoma antibodies against human milk fat globules recognize the I(Ma)
antigenic determinant: Galj81-4GlcNAc/81-6-. Carbohydr. Res. 120, 293-302.
360
J. E . P E N N I N G T O N , S. R A S T A N , D . R O E L C K E AND T. F E I Z I
GOOI, H. C., WILLIAMS, L. K., UEMURA, K., HOUNSELL, E. F., MCILHINNEY, R. A. J. & FEIZI, T.
(1983d). A marker of human foetal endoderm defined by a monoclonal antibody involves
Type 1 blood group chains. Molec. Immunol. 20, 607-613.
Gooi, H. C., VEYRIERES, A., ALAIS, J., SCUDDER, P., HOUNSELL, E. F. & FEIZI, T. (1984). Further
studies of the specificities of monoclonal anti-i and anti-I antibodies using chemically
synthesized, linear oligosaccharides of the poly-N-acetyllactosamine series. Molec. Immunol.
21, 1099-1104.
HAKOMORI, S. (1981). Blood group ABH and Ii antigens of human erythrocytes: chemistry,
polymorphism and their developmental change. Semin. Hematol. 18, 39-62.
HOUNSELL, E. F., GOOI, H. C. & FEIZI, T. (1981). The monoclonal antibody anti-SSEA-1
discriminates between fucosylated Type 1 and Type 2 blood group chains. FEBS Lett. 131,
279-282.
KABAT, E. A., LIAO, J., SHYONG, J. & OSSERMAN, E. F. (1982). A monoclonal IgM macroglobulin
with specificity for lacto-N-tetraose in a patient with bronchogenic carcinoma. /. Immunol.
128, 540-544.
KANNAGI, R., COCHRAN,N. A.,ISHIGAMI,F.,HAKOMORI,S., ANDREWS,P. W.,KNOWLES,B. B. &
SOLTER, D. (1983a). Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a
unique globo-series ganglioside isolated from human teratocarcinoma cells. EMBO J. 2,
2355-2361.
KANNAGI, R., LEVERY, S. B., ISHIGAMI, F., HAKOMORI, S., SHEVINSKY, L. H., KNOWLES, B. B. &
SOLTER, D. (19836). New globoseries glycosphingolipids in human teratocarcinoma reactive
with the monoclonal antibody directed to a developmentally regulated antigen, stage-specific
embryonic antigen 3. J. biol. Chem. 258, 8934-8942.
KAPADIA, A., FEIZI, T. & EVANS, M. J. (1981). Changes in the expression and polarization of
blood group I and i antigens in post-implantation embryos and teratocarcinomas of mouse
associated with cell differentiation. Expl Cell Res. 131, 185-195.
KNOWLES, R. W., BAI, Y., DANIELS, G. L. & WATKINS, W. M. (1982). Monoclonal anti-type 2 H:
an antibody detecting a precursor of the A and B blood group antigens. /. Immunogenet. 9,
69-76.
Li, Y-T. & Li, S-C. (1972). a-Galactosidase from figs. Methods in Enzymol. 28, 714-721.
LIMAS, C. & LANGE, P. (1982). A, B, H antigen detectability in normal and neoplastic
urothelium. Influence of methodologic factors. Cancer 49, 2476-2484.
MURAMATSU, T., GACHELIN, G., NICOLAS, J. F., CONDAMINE, H., JAKOB, H. & JACOB, F. (1978).
Carbohydrate structure and cell differentiation: Unique properties of fucosyl-glycopeptides
isolated from embryonal carcinoma cells. Proc. natnAcad. Sci., U.S.A. 75, 2315-2319.
QUINN, P., BARROS, C. & WHITTINGHAM, D. G. (1982). Preservation of hamster oocytes to assay
the fertilizing capacity of human spermatozoa. /. Reprod. Fert. 66, 161-168.
RASTAN, S., THORPE, S. J., SCUDDER, P., BROWN, S., GOOI, H. C. & FEIZI, T. (1985). Cell
interactions in preimplantation embryos: evidence for involvement of saccharides of the polyN-acetyllactosamine series. /. Embryol. exp. Morph. 87, 115-128.
SCUDDER, P., HANFLAND, P., UEMURA, K. & FEIZI, T. (1984). Endo-/S-galactosidases of Bacteroides
fragilis and Escherichia freundii hydrolyse linear but not branched oligosaccharide domains of
glycolipids of the neolacto series. /. biol. Chem. 259, 6586-6592.
SCUDDER, P., UEMURA, K., DOLBY, J., FUKUDA, M. N. & FEIZI, T. (1983). Isolation and
characterization of an endo-)3-galactosidase from Bacteroides fragilis. Biochem. J. 213,
485-494.
SOLTER, D. & KNOWLES, B. B. (1978). Monoclonal antibody defining a stage-specific mouse
embryonic antigen (SSEA-1). Proc. natnAcad. Sci., U.S.A. 75, 5565-5569.
STERN, P. L., WILLISON, K. R., LENNOX, E., GALFRE, G., MILSTEIN, C., SECHER, D., ZIEGLER, A. &
SPRINGER, T. (1978). Monoclonal antibodies as probes for differentiation and tumor-associated
antigens: a Forssman specificity on teratocarcinoma stem cells. Cell 14, 775-783.
G., EVANS, M. J., WILLISON, K. R. & STERN, P. L. (1981). Expression of Forssman
antigen in the post-implantation mouse embryo. /. Embryol. exp. Morph. 61, 117-131.
SZULMAN, A. E. (1980). The ABH blood groups and development. In Current Topics in
Developmental Biology 14, Part II (ed. M. Friedlander), pp. 127-145. London: Academic
Press.
STINNAKRE, M.
Saccharide structures of the mouse embryo
361
S. J. & FEIZI, T. (1983). Blood group antigens in the normal and neoplastic bladder
epithelium. /. din. Path. 36, 873-883.
UEMURA, K., CHILDS, R. A., HANFLAND, P. & FEIZI, T. (1983). A multiplicity of erythrocyte
glycolipids of the neolacto series revealed by immuno-thin layer chromatography with
monoclonal anti-I and anti-i antibodies. Biosci. Rep. 3, 577-588.
UEMURA, K., ROELCKE, D., NAGAI, Y. & FEIZI, T. (1984). The reactivities of human erythrocyte
autoantibodies anti-Pr2, anti-Gd, Fl and Sa with gangliosides in a chromatogram binding
assay. Biochem. J. 219, 865-874.
THORPE,
VOAK, D., LENNOX, E., LOWE, A. D.,DOWNIE, D. M., MERRY, A. H., JARVIS, J. &MILSTEIN,C.
(1983). Principles of monoclonal antibodies and their use for ABO and antiglobulin reagents.
Proc. Brit. Blood Transf. Soc. 1, 83-112.
WHrrnNGHAM, D. G. (1971). Culture of mouse ova. /. Reprod. Fert. (suppl.) 14, 7-21.
WILLISON, K. R., KAROL, R. A., SUZUKI, A., KUNDU, S. K. & MARCUS, D. M. (1982). Neutral
glycolipid antigens as developmental markers of mouse teratocarcinoma and early embryos:
an immunologic and chemical analysis. /. Immunol. 129, 603-609.
WOOD, C. W., KABAT, E. A., MURPHY, L. A. & GOLDSTEIN, L J. (1979). Immunochemical studies
of the combining sites of the two isolectins. A4 and B4 isolated from Bandeiraea simplificolia.
Arch. Biochem. Biophys. 198, 1-11.
Wu, T. C , WAN, Y-J. & DAMJANOV, I. (1983). Fluorescein-conjugated Bandeiraea simplicifolia
lectin as a marker of endodermal, yolk sac and trophoblastic differentiation in the mouse
embryo. Differentiation 24, 55-59.
(Accepted 16 July 1985)