/ . Embryo!, exp. Morph. Vol. 57, pp. 25-36, 1980
Printed in Great Britain © Company of Biologists Limited 1980
25
Changes in fucosyl-glycopeptides during early
post-implantation embryogenesis in the mouse
By TAKASHI MURAMATSU, HUBERT CONDAMINE,
GABRIEL GACHELIN 1 AND FRANQOIS JACOB
From the Service de Genetique Cellulaire du College de France
and the Institut Pasteur, Paris
SUMMARY
Six-day to 12-day mouse embryos were dissected and radiolabelled by culture in the presence
of [3H]fucose. The radiolabelled embryos were extensively digested with Pronase. The
resulting glycopeptides were analysed by Sephadex G-50 column chromatography. Glycopeptides from 6-day-old embryos were separated into two main peaks: one eluted near the
excluded volume, the other in a well-retarded position. This elution profile was similar to that
observed with glycopeptides prepared from embryonal carcinoma cells. The relative amount
of the high-molecular-weight glycopeptides decreased during embryonic development and
particularly around day 9. Glycopeptide elution profiles from 10-day embryos, or isolated
organs of 12-day embryos, were indistinguishable from those obtained from differentiated
teratocarcinoma-derived or adult cells. At least 30% of the large molecular weight glycopeptides appear to be located at the cell surface.
INTRODUCTION
The involvement of specific cell-cell interactions in differentiation sequences
has often been postulated (Bennett, Boyse & Old, 1971). The evidence, however,
for the existence of cell surface components whose function would be to mediate
such effects is rather scarce. This problem has been approached in our laboratory
by looking for cell surface antigens from undifferentiated embryonal carcinoma
(EC) cells, which would disappear, or be altered, as differentiation of these cells
proceeds, and thus be likely candidates to play a role in differentiation (reviewed
in Jacob, 1977, and Gachelin, 1978). More recently, we have studied membranebound carbohydrates of EC cells, and we have reported the presence in these of
large amounts of unusual fucosyl-glycopeptides, characterized by their apparent
molecular weight exceeding 7000 daltons, as estimated by gel filtration of the
fucose-labelled glycopeptides (Muramats.i et al. 1978). Further chemical
analysis has shown that: (a) they are neither glycolipids, nor mucopolysaccharides, nor mucine-type-glycopeptides with short oligo-saccharide chains,
nor products of incomplete digestion (Muramatsu et al. 1979 #); (b) they all
1
Author's address: Institut Pasteur, 25 rue du Dr. Roux 75015 Paris, France.
26
T. MURAMATSU AND OTHERS
have the 'Glue NAQ?
>Gal sequence' as internal core structure (Muramatsu
et al. 19796, c); (c) they derive in part from cell surface components (Muramatsu et al. 1919 b, c; Prujansky et al. 1979).
Fucosyl-glycopeptides of high molecular weight were not detected in the
various differentiated cell types studied, and they were found to disappear from
EC cells as these differentiated in vitro (Muramatsu et al. 1978). Since EC cells
are currently used as a model for the study of normal embryonic cell differentiation (Graham, 1977), it was important to determine whether glycopeptides of
high molecular weight are present in normal embryos: it has been reported that
the elution profile of glycopeptides prepared from [3H]fucose-labelled preimplantation embryos was similar to that of EC cells (Muramatsu et al. 1978).
In the present paper, we report that elution profiles of fucosyl-glycopeptides
obtained from 6- to 10-day embryos are progressively altered. By day 10 the
ratio of fucose incorporated into high and low molecular weight glycopeptides
reaches a value characteristic of normal adult cells. In addition, we present
evidence that at least part of the fucosyl-glycopeptides of early post-implantation cells are located on the surface of (or between) embryonic cells.
MATERIALS AND METHODS
(1) Embryo dissection and culture
C57B1/6 or ¥x (C57Bl/6xCBA) females were penned with Fx (C57B1/6
x CBA) males and checked daily for the presence of a vaginal plug. The day
when a vaginal plug was detected was taken as day 0 of pregnancy. Eight-day
and 9-day embryos were dissected as described elsewhere (Buc-Caron, Condamine & Jacob, 1978). Six-day and 7-day embryos were dissected free from the
decidua and trophoblast, but retained most of their parietal yolk sac and extraembryonic ectoderm. Whole 6- to 8-day embryos and crudely minced older
embryos were cultured for 6 h at 37 °C under an atmosphere of 12 % CO2 in
air, in 1-2 ml of Dulbecco's modified Eagle's medium containing 15 % fetal
calf serum (Gibco) and 20 /tCi/ml of L-[3H]fucose (10-20 Ci/niM, CEA, France).
(2) Preparation and analysis of embryonic cell extracts
The labelled embryos were washed three times with Hanks balanced salt
solution and processed. Pronase digestion of labelled embryos was carried out as
described previously (Muramatsu et al. 1978): the incubation volume was 1 ml
for 6- and 7-day embryos, and 2 ml in the other cases. The digest was applied
on top of a column of Sephadex G-50 superfine (1-7 x 60 cm) equilibrated and
eluted with a pH 6-0, 0-05 M ammonia-acetic acid buffer; 2-3 ml fractions were
collected. Radioactivity in the fractions was determined by liquid scintillation
counting. Standard glycopeptides for column calibration were as described
previously (Muramatsu, Ogata & Koide, 1976).
Results are expressed as elution profiles, i.e. as radioactivity counts (in cpm)
Fucosyl-glycopeptides in mouse embryogenesis
27
recovered in each fraction eluted from the column, plotted against fraction
number. The curves thus obtained generally presented two main peaks corresponding to glycopeptides of relatively high and low molecular weight. In order
to characterize these elution profiles more quantitatively, the ratio of counts
recovered in low-molecular-weight peak to counts recovered in the highmolecular-weight peak, was used. This ratio should not be considered, however,
as reflecting genuine quantification, since little is known of the structure or of
turn over of molecules under study (see Discussion).
Identification of radioactively labelled saccharides was carried out by paper
chromatography of acid-hydrolysis products, as described previously (Muramatsu, Atkinson, Nathenson & Ceccarini, 1973).
(3) Release offucosyl-glycopeptides by mild trypsinization
Eight- to 10-day embryos were labelled as usual, washed carefully by three
successive passages in the pre-warmed Hanks medium and placed in a saline
trypsin solution (NaCl 8 g/1, KC1 0-4 g/1, glucose 1 g/1, NaHCO 3 0-5 g/1,
trypsin 0-5 g/1, EDTA 0-2 g/1) at 37 °C. After about 10 min the embryos were
gently and repeatedly pipetted through a capillary pipette. Trypsin action was
allowed for 3 more minutes, then stopped by adding an equal volume of proteinrich medium (Eagles medium+15 % foetal calf serum). The now-dissociated
cells were centrifuged gently in a Beckman microfuge (450 #, 4 min). The
supernatant was collected, cleared of debris by centrifugation in a Beckman
Airfuge (120000 #, 10 min) and digested with pronase. The pellet of cells was
resuspended in Eagles medium+15% foetal calf serum. Viable cells were
estimated on an aliquot using the Trypan blue exclusion test (Artzt et al. 1973).
The remaining cells were digested with Pronase.
RESULTS
(1) Fucosyl-glycopeptides from total embryos
Six-day embryos were dissected free from their decidua and trophoblast, but
retained most of their parietal yolk sac and extra-embryonic ectoderm. After
6 h of culture in the presence of [3H]fucose, the mean incorporation of radioactive fucose into water-soluble glycopeptides was about 400 cpm per 6-day
embryo, with a rather large variability probably due to the variability of developmental state and cell number of embryos. Pools of embryos (5-10) were analysed.
The soluble glycopeptides of 6-day embryos were separated by Sephadex G-50
column chromatography into two main classes: one class is made of glycopeptides of large molecular weight eluted near the excluded volume, whose apparent
molecular weight is higher than 7000 daltons; the other class comprises glycopeptides eluted in well-retarded position, with a molecular weight around
2500 daltons, a value based on the calibration of the column with standard
glycopeptides (see legend to Fig. 1). This elution profile (Fig. 1A) appears to be
28
T. MURAMATSU AND OTHERS
1
1
1
I
-\
A
ft
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v0 %..••...•••
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Fraction number
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50
Fucosyl-glycopeptides in mouse embryogenesis
29
3
very similar to the one obtained with both [ H]fucose-labelled EC cells and
preimplantation embryos (Muramatsu et al. 1978).
Unlike 6- or 7-day embryos, 8-day and older embryos were dissected out of
their parietal yolk sac. Seven- and 8-day embryos were incubated for 6 h in
culture medium plus [3H]fucose, as above, without further treatment. In
contrast, older embryos were cut into five to eight pieces, in order to facilitate the
diffusion of metabolites through the embryonic tissues. Again, pooled embryos
were used. The mean incorporation of [3H]fucose per embryo into soluble
glycopeptides rapidly increased with embryonic age (from 6000 cpm per 7-day
embryo, to 80 000 cpm per 10-day embryo). When fucosyl-glycopeptides prepared from 7- or 8-day embryos were analysed by Sephadex G-50 column
chromatography, a biphasic elution profile was again observed. However, a
slight decrease in the relative amount of high-molecular-weight glycopeptides
was noticeable (Fig. IB, C). The decline in the relative amount of high molecular
weight material was clearly detectable in 9-day embryos (Fig. ID), and even
more evident in 10-day embryos: at that stage, the elution profile was essentially
similar to that from fully differentiated cell types (Fig. IF) (Muramatsu et al.
1978).
In another set of experiments, pooled 9-day-old embryos were incubated in the
culture medium containing [3H]fucose for 24 h instead of the standard 6 h
period. Under these conditions, no morphogenesis occurred but cells proliferated and differentiated. The elution profile (Fig. IE) of glycopeptides recovered
in that case was now similar to that obtained from 10-day embryos. In previous
experiments, such a longer incubation time was found not to alter the glycopeptide elution profile from preimplantation embryos (Muramatsu et al. 1978),
and also from EC cells (Gachelin, unpublished result).
These results thus show that although the total amount of radioactive fucose
incorporated in 6 h into high-molecular-weight fucosyl-glycopeptides per
FIGURE 1
Sephadex column chromatography of [3H]fucose-labelled glycopeptides synthesized
by mouse embryos at various stages. The procedure is described in Materials
and Methods. The totality of pronase solubilized material was applied on top
of the column. Positions of standard substances were as follows: blue dextran
(fractions 22-24); [14C]acetylated fetuin glycopeptides (M.W. 3500, fractions 27-39);
[14C]acetylated IgG glycopeptides (M.W. 2000, fractions 45-47); fucose (M.W. 164,
fractions 60-63). Results are expressed as radioactivity recovered in each fraction
versus fraction numbers. Scales have been normalized so as to give the low molecular
weight peak the same height.
(A) radioactivity recovered from 6-day embryos (6 embryos)
(B) radioactivity recovered from 7-day embryos (21 embryos)
(C) radioactivity recovered from 8-day embryos (16 embryos)
(D) radioactivity recovered from 9-day embryos (5 embryos)
(E) radioactivity recovered from 9-day embryos cultured for 24 hours (7 embryos)
(F) radioactivity recovered from 10-day embryo? (3 embryos).
57
30
T. MURAMATSU AND OTHERS
2000
1000
30
40
50
Fraction number
60
Fig. 2. Sephadex G-50 column chromatography (30 x 1 cm) of [3H]fucose-labelled
glycopeptides synthesized by parietal yolk-sac cells from 8-day embryos. Blue
dextran was eluted in fractions 32-34, IgG glycopeptides in fractions 65-67, and
fucose in fractions 77-78.
embryo greatly increases between days 6 and 10 of embryogenesis (from 120 cpm
per 6-day embryo to c. 2000 per 10-day embryo), there is at the same time a 10fold decrease in the ratio of fucose incorporated into high- and low-molecularweight glycopeptides respectively, this decrease being particularly striking
between day 9 and day 10. The behaviour of these two classes of glycopeptides
during post-implantation embryogenesis is thus very reminiscent of what has
been previously observed during in vitro differentiation of EC cells (Muramatsu
et al. 1978).
(2) Fucosyl-glycopeptides from isolated embryonic or extra-embryonic organs
As has been noted above, 8-day and older embryos were incubated free from
their parietal yolk sac, unlike 6- and 7-day embryos. Since after day 8 of embryogenesis, a steady decrease in the relative incorporation of radioactive fucose
into high-molecular-weight glycopeptides is observed, one could wonder
whether incorporation was taking place in yolk-sac cells of 8-day and older
31
Fucosyl-glycopeptides in mouse embryogenesis
5000 -
a 10000 -
2000 -
50
Fraction number
Fig. 3. Sephadex G-50 column chromatography of [3H]fucose-labelled glycopeptides
synthesized by dissected embryonic organs. Same column as in Fig. 1. (A) Radioactivity recovered from 10-day embryo head part (10 embryos). (B) Radioactivity recovered from 12-day embryo head part (6 embryos). (C) Radioactivity recovered
from 12-day embryo liver (4 embryos).
embryos at a high rate. Therefore, parietal yolk sacs from 8-day embryos were
dissected apart and incubated under standard conditions, digested with Pronase
extensively, and the resulting soluble glycopeptides were analysed by Sephadex
G-50 column chromatography. Yolk-sac cells were found to incorporate substantial amounts of radioactive fucose (c. 104 cpm incorporated per 8-day yolk
sac). The fucosyl-glycopeptides obtained from them gave an elution profile
essentially similar to that from differentiated cells (Fig. 2), with about ten times
more radioactive fucose recovered into glycopeptides of low than high molecular
weight. It seems therefore unlikely that the relative decrease of fucose recovery
into high-molecular-weight glycopeptides observed in 8-day and older embryos
is due to these embryos being incubated free from their yolk sac.
3-2
32
T. MURAMATSU AND OTHERS
Similarly, a few organs from 10- and 12-day embryos were incubated apart to
check whether cell populations might remain in some embryonic compartments
which would preferentially incorporate fucose in high-molecular-weight
glycopeptides at a time when the bulk of the embryo incorporates fucose in
low-molecular-weight material preferentially. Head parts from 10-day embryos,
brain and liver from 12-day embryos were thus allowed to incorporate radioactive fucose, Pronase digested and their fucosyl glycopeptides analysed in the
usual way. The elution pattern obtained in all three cases (Fig. 3 A, B, C) was
again found to be very similar to the one yielded by teratocarcinoma-derived
differentiated cells, with an amount of radioactivity incorporated into lowmolecular-weight material clearly much higher than in the high-molecularweight peak.
(3) Release of fucosyl-glycopeptides from embryonic cells following mild trypsin
digestion
In order to determine whether the fucosyl-glycopeptides synthesized by early
embryonic cells are at least partly located on the outer part of cell membranes,
embryos which had incorporated radioactive fucose were submitted to mild
tryptic digestion under conditions in which most of the cells were shown to be
viable. Eight-day embryos were chosen for these experiments because small
batches of embryos of this age have suitable levels of radioactive incorporation
and the relative amount of labelling into the high-molecular-weight material is
still rather high. Eight-day embryos submitted to trypsinization for about 10
min (see Materials and Methods) yielded well-dissociated cell suspensions with a
few aggregates of less than ten cells. After the treatment about 10% of the cells
were scored dead in the trypan blue exclusion test. In contrast, 30-40 % of total
radioactivity incorporated into non-dialysable material was recovered in the
trypsinized cell supernatant. When this soluble material, as well as the radioactive material remaining associated with the cells, were digested by Pronase,
and then analysed by Sephadex G-50 column chromatography, both preparations essentially gave the same elution profiles. No enrichment in either lowor high-molecular-weight material was detected in the trypsin-released fraction
(Table 1). These results are very similar to those obtained with both murine
EC cells (Gachelin, unpublished) and human EC cells (Muramatsu et al. 1979 a).
Taken together, these results suggest that at least one third of all fucosyl
glycopeptides synthesized by 8-day embryos are present on the surface of
embryonic cells, or between embryonic cells.
(4) Recovery of ^H]fucose from 8-day embryo fucosyl-glycopeptides
Fucose is known to yield few transformation products following its uptake by
adult cells (Kaufman & Ginsburg, 1968; Buck, Glick & Warren, 1970; van
Beek, Smets & Emmelot, 1973), or by embryonal carcinoma cells (Muramatsu
et al. 1978). In order to check whether this was also the case with normal early
Fucosyl-glycopeptides in mouse embryogenesis
33
Table 1. Release offucosyl-glycopeptides from 8-day embryonic
cells following mild trypsinization
Experiment 1
(8 embryos)
Experiment 2
(15 embryos)
Radioactivity recovered in:
Trypsinized cell supernatant
Total glycopeptides
77,300
181,000
High-molecular-weight glycopeptides
11,400
32,000
Low-molecular-weight glycopeptides
43,000
121,000
Trypsinized cell pellet
Total glycopeptides
102,000
390,000
High-molecular-weight glycopeptides
12,400
55,000
49,600
226,000
Low-molecular-weight glycopeptides
Dead/live cells after trypsinization
8/219(3%)
24/190(11 %)
3
Eight-day embryos were dissected, incubated with [ H]fucose and trypsinized as indicated in
Materials and Methods. Trypsinized cell supernatant and pellet were dialysed and then
extensively digested with pronase; the resulting digest was counted (total GP) and then submitted to Sephadex G-50 column chromatography analysis. Radioactivity is expressed in
cpm. High- (or low-) molecular-weight fucosyl-glycopeptide radioactivity is the sum of counts
found in the high- (or low-) molecular-weight peak fractions (see legend to Table 2 for the
definition of these fractions).
embryonic cells, [3H]fucose-labelled 8-day embryos were hydrolysed by HC1
in the presence of unlabelled sugar. Products from hydrolysis were separated
by paper chromatography, and the monosaccharides localized. The chromatogram was cut into pieces and the radioactivity associated with each section
counted. All the radioactivity recovered in monosaccharides was found to
co-migrate with fucose.
DISCUSSION
It has been shown previously that embryonal carcinoma cells and preimplantation embryos both share distinctive glycopeptides of unusual physicochemical properties (Muramatsu et al. 1978, 1979a, b, c). When embryonal
carcinoma cells are allowed to differentiate in vitro, one observes a progressive
decrease in the contribution of these high-molecular-weight glycopeptides
and a correlative increase in a heretofore minor glycopeptide family, defined by
a lower molecular weight: when the differentiation process has been completed,
more than 90 % of the [3H]fucose was recovered into this low-molecular-weight
component.
The results reported in this paper indicate that a similar shift in the molecular
'landscape' of embryonic cells occurs after day 8 of embryogenesis, which
seems to be the normal counterpart of the phenomenon observed with embryonal carcinoma cells first. These results are summarized in Table 2, where the
ratios of radioactive fucose incorporated into low-molecular-weight glyco-
34
T. MURAMATSU AND OTHERS
Table 2. Relative [sH]fucose incorporation in low- and high-molecular-weight
glycopeptides in various cell lines and embryos at various developmental age
Cell types
F9
PCC3/A/1
PCC4 aza
PCC3/A/1-28
Pys-2
Fibroblasts 129
Lymphocytes 129
Description and references
Nullipotent EC (Bernstine et al. 1973)
Multipotent EC (Jakob et al. 1973)
Multipotent EC resistant to azaguanine (Jakob
et al. 1973)
Fully differentiated PCC3/A/1 (Nicolas et al.
1975)
Endodermal carcinoma (parietal yolk-sac carcinoma) (Lehman et al. 1974)
Newborn 129/Sv
fibroblasts
Normal adult lymph node lymphocytes
Low/high ratio
1 -7
1-7
1-7
26-7
15-0
22
25
Embryos
Total:
3-day
6-day
7-day
8-day
9-day
10-day
Isolated organs:
8-day
10-day
12-day
12-day
1 7
1-9
2-4
3-2
5-1
22
Parietal yolk sac
Head part
Head part
Liver
11-2
18
26
18
The low/high ratio represents the ratio between the counts recovered in fraction 35-50
('low', Figs. 1 and 2) and the counts recovered in fraction 21-28 ('high', Figs. 1 and 2) or
their equivalent in other columns. Some of the data are taken from results already published
as elution profiles (Muramatsu et al. 1978).
peptides towards radioactive fucose incorporated into high-molecular-weight
glycopeptides at various embryonic ages are presented. From day 6 to day 10
there is a 10-fold increase in this ratio, so that, by this time the low-molecularweight glycopeptides are obviously largely predominant as they will remain
afterwards.
The causal relation, if any, which these changes may have with differentiation
processes occurring at this time, remains to be found. In looking for it, two facts
should be kept in mind. First, some evidence is presented here that the molecular
species under study are largely located on the surface of, or between, 8-day
embryonic cells. Secondly, the changes in the relative abundance of glycopeptides of high molecular weight are readily apparent at a time of embryogenesis when the early embryonic cell surface marker F9 antigen ceases to be
detected on embryonic cell surfaces (Buc-Caron et al. 1978). We have recently
shown that high-molecular-weight glycopeptides are associated with the various
Fucosyl-glycopeptides in mouse embryogenesis
35
components of the F9 antigen isolated from EC cells (Muramatsu et al. 1979 b),
and early embryonic cells (Gachelin, unpublished data): the results represented
here point further to a similarity in the evolution of these two classes of EC cell
characteristic molecules. It is therefore tempting to interpret these data as
meaning that dramatic changes in polysaccharide metabolism occur during days
7-9 of mouse embryogenesis.
However, other interpretations are compatible with the data presented here.
As an example, the subcellular location as well as the turnover rate of the highand low-molecular-weight glycopeptides could be different: low-molecularweight glycopeptides, such as those associated to the LETS protein (Carter &
Hakomori, 1979), could accumulate in extracellular spaces, whereas highmolecular-weight glycopeptides would be permanently turned over. One would
thus observe a decrease in the relative contribution of high-molecular-weight
species. A more trivial explanation, where the reduction of high-molecularweight glycopeptides in 9- and 10-day embryos would be due to radioactive
fucose having no free access to some cellular compartments, seems unlikely,
because autoradiographs performed on 10-day-embryo sections did not, in our
hands, reveal any limitation to the diffusion of fucose among the various
embryonic tissues (Condamine & Gaillard, unpublished result). A detailed
structural analysis of these glycopeptides, which would allow the study of
specific transferases involved in their synthesis, seems to be the only way leading
to a more definitive interpretation of the phenomenon reported above.
We thank Francoise Kelly for a critical reading of the manuscript.
T.M. has been on leave of absence from Kobe University School of Medicine, and was
supported by the Franco-Japanese exchange programme between l'lnstitut National de la
Sante et de la Recherche Medicale and the Japan Society for the Promotion of Science. This
work was supported by grants from the Centre National de la Recherche Scientifique, the
Institut National de la Sante et de la Recherche Medicale, the Delegation Generate a la
Recherche Scientifique et Technique, the National Institute of Health, and the Andre Meyer
Foundation.
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(Received 15 May 1979, revised 20 December 1979)