/. Embryol. exp. Morph. Vol. 43, pp. 147-156, 1978
Printed in Great Britain © Company of Biologists Limited 1978
147
Localization of trophoblast-defined surface antigens
during early mouse embryogenesis
By R. F. SEARLE 1 AND E. J. J E N K I N S O N 1
From the Reproductive Immunology Research Group,
Department of Pathology, The Medical School, University of Bristol
SUMMARY
The binding pattern of a rabbit antiserum raised against mouse ectoplacental-cone trophoblast on component cell populations in the pre-implantation and early post-implantation
mouse embryo has been examined at the electron-microscope level using an immunoperoxidase-labelling technique. Binding was not detectable on the 1-cell stage, appeared at low
levels at the 8-cell stage and was heavy on the trophectoderm and its trophoblast giant cell
and extra-embryonic ectoderm descendants in the post-implantation embryo. In contrast,
immunosurgically isolated 3i-day inner cell masses (ICM) showed only slight labelling,
whilst ICM derivatives in the 7^-day embryo were unlabelled.
The results indicate that the antiserum may be identifying a trophoblast-specific surface
determinant(s), which appears with the differentiation of the trophectoderm and is maintained on some of the cell populations derived from this tissue at least until the early postimplantation stages.
INTRODUCTION
Information on the composition and organization of embryonic cell surfaces
is relevant both to an understanding of differentiation at the membrane level
and to the analysis of cellular interactions during embryogenesis. However,
apart from fine-structural studies (Calarco & Brown, 1969; Potts, 1969;
Nilsson, 1974; Ducibella, Albertini, Anderson & Biggers, 1975; Ducibella &
Anderson, 1975) and observations on cell movement and behaviour (Mulnard,
1967), it is only recently that early mammalian development has been considered in this light.
Attempts to investigate the surface of the pre-implantation mouse embryo
have so far included biochemical analysis of enzyme-stripped surface components (Pinsker & Mintz, 1973), electron-histochemical (Nilsson, Lindqvist &
Ronquist, 1973, 1975) and combined electron-histochemical/enzyme-stripping
studies (Jenkinson & Searle, 1977), examination of lectin binding (Enders &
Schlafke, 1974; Schlafke & Enders, 1975; Johnson, Eager, Muggleton-Harris
& Grave, 1975; Rowinski, Solter & Koprowski, 1976; Sellens, 1976) and the
use of labelled antisera raised against histocompatibility and other antigens
1
Authors'1 address: Reproductive Immunology Research Group, Department of Pathology
The Medical School, University of Bristol, Bristol BS8 1TD, U.K.
148
R. F. SEARLE AND E. J. J E N K I N S O N
(see Jenkinson & Billington, 1977). The present study concerns the production
of an antiserum to mouse ectoplacental-cone trophoblast (EPC) and examination
of the pattern of labelling obtained with this antiserum on some of the component cell populations of the pre- and early post-implantation embryo using an
immunoperoxidase labelling technique.
MATERIALS AND METHODS
Antiserum
Pregnant females from a randomly breeding colony derived from C57BLxA
hybrid mice were killed 1\ days after the finding of a vaginal plug (day of
plug = day 0), the decidual swellings removed from the uterine horns and the
intact embryos isolated as described by Kirby (1971). After separation from
their embryonic sacs and removal of any adherent Reichert's membrane,
groups of 50-75 ectoplacental cones (EPCs) were washed several times in
Dulbecco's phosphate-buffered saline (PBS), emulsified in Freund's complete
adjuvant and injected intramuscularly into a New Zealand White rabbit.
Following three such injections, at 4-6 weekly intervals, the rabbit was testbled and given a booster dose of 40 EPCs without adjuvant. Antiserum was
collected 10 days later, tested for cytotoxicity on adult mouse lymph-node cells
(titre 1/128), divided into aliquots and stored frozen at - 2 0 °C until required.
Prior to use, thawed aliquots were heat-inactivated and absorbed repeatedly
with washed, packed mouse spleen cells for 1 h at 37 °C at 2:1 volume ratio
until complement-mediated cytotoxicity to adult mouse lymph-node cells was
abolished. A further two absorptions were carried out before the antiserum
was employed in the immunoperoxidase test.
Normal rabbit serum for use in control experiments was subjected to a similar
absorption procedure.
Embryonic material
Spontaneously ovulating, randomly bred female mice provided material at
several stages of pre- and post-implantation embryonic development. All
embryos were recovered and handled in PBS supplemented with 2 % bovine
serum albumin (BSA).
Pre-implantation embryos. Unfertilized eggs were recovered on day 0 following
mating with a vasectomized male. Fertilized 1-cell and 8-cell embryos from
normal matings were collected from the oviducts on days 0 and 2 respectively;
morulae and blastocysts were recovered from the uterus during day 3.
Removal of the zona pellucida from all embryos was accomplished by partial
digestion in 0-5 % pronase in PBS followed by gentle mechanical disruption in
a glass micropipette.
Immunosurgically isolated inner cell masses. Inner cell masses (ICMs) from
3^-day blastocysts were isolated by a procedure based on that of Solter &
Trophoblast-defined
surface antigens on mouse embryos
149
Knowles (1975). Zona-free blastocysts were incubated for 1 h at 37 °C in a
1 : 20 dilution of rabbit anti-mouse antiserum in medium RPMI 1640 + 10%
foetal calf serum (FCS), washed in PBS and incubated for a further 30 min in
agar-absorbed guinea-pig serum at a final dilution of 1:9. After careful washing
the lysed trophectoderm cells were dislodged by gentle pipetting in a narrow
bore glass micropipette. Isolated ICMs were allowed to recover for 1 h in
FCS-supplemented RPMI before use.
Peri-implantation embryos. Blastocysts in the immediate pre-implantation
phase were obtained by flushing the uteri of ovariectomized, progesteronemaintained, pregnant females 14-18 h after the administration of 0-1 /*g of
17-/? oestradiol (Yoshinaga & Adams, 1966). Blastocyst outgrowths in vitro
were produced by culturing 3^-day blastocysts for 3 days in 0-2 ml volumes of
RPMI + 1 0 % FCS in the wells of Cooke microtitre plates under a humidified
gas phase of 5 % C0 2 in air.
Post-implantation embryos. Post-implantation embryos were recovered early
on day 7 and separated into embryonic sacs and EPCs as outlined above. For
examination of the labelling characteristics of the outer proximal endoderm
layer, embryonic sacs were left intact. Examination of the inner components
was carried out following removal of this outer layer, which was achieved by
drawing the isolated sacs in and out of a flame-polished pipette of a diameter
slightly narrower than their transverse axis after a 15 min exposure to 1%
trypsin (Difco 1:250) at 37 °C. In this way the endoderm with its basement
membrane could be unrolled over the inner portion of the sac, leaving a core
consisting of an upper region of extra-embryonic ectoderm and a lower region
of embryonic ectoderm/mesoderm. Electron microscopical examination of such
preparations revealed the complete removal of the endoderm and its basement
membrane.
Immunoperoxidase labelling. This was performed by an indirect labelling
method (Searle et al. 1976). Embryonic material was incubated in the rabbit
anti-EPC antiserum at a dilution of 1:4 for 1 h, followed by a further 1 h
incubation in a peroxidase-conjugated IgG fraction of goat antiserum to rabbit
IgG (Miles Research Products) at a dilution of 1:10. All processing was carried
out on ice in the presence of 0-02 M sodium azide to minimize pinocytotic
uptake. Wherever possible, embryos from different stages were processed on
the same occasion and in the same vessel, especially when comparisons of
staining levels were to be made as in the case of isolated ICMs and intact
blastocysts. In all cases control embryos at equivalent stages were incubated in
absorbed normal rabbit serum and processed in parallel.
Electron microscopy. For electron microscopical examination, pre-implantation embryos were embedded in 2 % molten agar at 37 °C and, after cooling,
cut out in small blocks for ease of handling. These, together with the blastocyst
outgrowths in wells and the isolated fragments of post-implantation embryos,
were embedded in Epon and processed and sectioned as described previously
(Searle et al 1976).
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R. F. SEARLE AND E. J. J E N K I N S O N
Table 1. Immunoper oxidase labelling of mouse embryos by anti-EPC
antiserum
Labelling pattern*
Embryonic material
Unfertilized egg
Fertilized egg
8-cell embryo
16 to 32-cell embryo
3i-day blastocyst
Trophectoderm
Isolated ICM
Activated blastocyst
Blastocyst outgrowth in vitro : trophoblast
7^-day embryo
EPC trophoblast
Proximal endoderm
Embryonic ectoderm/mesoderm
Extra-embryonic ectoderm
—
-
++
++++
++++
+
+ + +/ + + + +t
++++
++++
+ + +
+ + + +, Heavy continuous labelling. + + +, Heavy discontinuous labelling. + +,
Widely spaced patches of label. +, Occasional patches of label. —, No labelling.
t Some cells slightly less heavily labelled.
* Assessment based on examination of 3-10 embryos.
RESULTS
The results, summarized in Table 1, indicate a changing pattern of expression
of the antigen(s) defined by the anti-EPC antiserum and demonstrate the presence of different degrees of labelling on the trophectoderm and inner cell mass
of the blastocyst as well as on some of the derivatives of these two cell populations.
In all cases control embryos or embryo fragments incubated in normal
rabbit serum before exposure to peroxidase-labelled goat anti-rabbit IgG were
completely unlabelled.
Pre-implantation embryos
Both the unfertilized ovum and the fertilized 1-cell embryo showed no
evidence of labelling after treatment with anti-EPC antiserum. By the 8-cell
stage, however, patches of labelling were observed on the outer aspect of each
blastomere and this increased markedly by the 16-32-cell stage to form a
continuous, heavy labelling over the outer surface of the morula and on the
trophectoderm of the blastocyst (Fig. 1).
Examination of immunosurgically isolated ICMs from 3^-day blastocysts
revealed only occasional patches of label on some cells (Fig. 2), in marked
contrast to the heavy labelling on the trophectoderm at this stage.
Trophoblast-defined surface antigens on mouse embryos
Fig. 1
151
Fig. 2
Electron-micrographs of immunoperoxidase labelling with anti-EPC antiserum
On pre- and post-implantation mouse embryos ( x 40000).
Fig. 1. Heavy membrane labelling characteristic of 3^-day trophectoderm and
trophoblast giant cells of the peri- and post-implantation embryo.
Fig. 2. Isolated patch of labelling occasionally seen on the surface of immunosurgically
isolated 3^-day ICM cells, which are otherwise devoid of electron-dense membrane
deposits.
Peri-implantation embryos
Blastocysts recovered from pregnant, ovariectomized, progesterone-maintained females 14-18 h after oestrogen administration, at a time when histocompatibility antigens are lost from the trophectoderm surface (Hâkansson,
Heyner, Sundqvist & Bergstrom, 1975; Searle et al. 1976), showed no evidence
of a decline in the expression of the determinants recognized by the anti-EPC
antiserum. Similarly, trophoblast giant cells in blastocyst outgrowths in vitro
showed heavy continuous electron-dense deposits on the cell membrane (Fig. 1).
Examination of the central knot of ICM cells in these outgrowths was not
possible since these did not withstand the repeated medium changes involved
in processing and embedding.
152
R. F. SEARLE AND E. J. JENKINSON
Fig. 3
Fig. 4
Fig. 3. Marked but discontinuous labelling on 7£-day extra-embryonic ectoderm.
Fig. 4. Absence of labelling on 7^-day embryonic ectoderm region cells after exposure
to anti-EPC antiserum. The absence of membrane deposits is also typical of other
non-labelling cell populations and of control tissues exposed to normal serum.
Post-implantation embryos
Since only the outer cells of the intact EPC are likely to be adequately exposed
to the antiserum, examination was confined to the peripheral trophoblast
giant cells, which showed a continuous pattern of surface labelling (Fig. 1)
similar to that on the trophectoderm of the blastocyst. In agreement with
experimental studies suggesting that the extra-embryonic ectoderm has a
trophectodermal origin (Gardner & Papaioannou, 1975), marked membrane
deposits were also observed on this tissue (Fig. 3), although labelling was not
continuous over the cell surface as in the case of the trophoblast. Conversely,
the proximal endoderm cells of the intact embryonic sac and the cells of the
embryonic ectoderm region of the endoderm-denuded sacs, both of which are
ICM-derived (Gardner & Papaioannou, 1975), were unlabelled (Fig. 4).
Overall, the differential staining pattern observed on the various components
of the embryo excludes the possibility that the reactivity of the antiserum is
due to any residual anti-species activity. The time of appearance, distribution
Trophoblast-defined
surface antigens on mouse embryos
153
and degree of labelling combine to indicate that it is primarily trophoblastspecific in its binding.
DISCUSSION
The use of labelled antisera as membrane probes has led to the identification
of a variety of determinants on the surface of the pre-implantation mouse
embryo. Although the relationship, and in some cases possible mutual identity,
of these components has yet to be elucidated, a comparison of their ontogeny
with that of the antigens detected by the anti-EPC antiserum suggests that the
latter do not correspond to any of the immunologically defined surface products
previously described.
Guinea-pig antiserum to the so-called mouse ' egg-antigen' detects components
on unfertilized and 1-cell stage embryos which are absent from the early postimplantation trophoblast (Baranska, Koldovsky & Koprowski, 1970 ; Moskalewski & Koprowski, 1972), whilst heterologous antiserum raised against mouse
placentae also gives positive reactions with unfertilized and 1-cell embryos
(Wiley & Calarco, 1975). The antigens detected by the anti-EPC antiserum, on
the other hand, are not detectable on the unfertilized egg and 1-cell embryo,
but are weakly expressed at the 8-cell stage and increase to give strong staining
on the morula which persists on the trophectoderm of the blastocyst and on
the post-implantation trophoblast giant cells. This pattern also differs from
that observed with autologous antiserum to F-9 teratoma antigens (Artzt et al.
1973) and heterologous antiserum to whole mouse blastocysts (Wiley & Calarco,
1975), both of which show peak levels of reactivity at the 8-12 cell stage with
a subsequent decline. Similarly, mutual identity of the EPC-defined antigens
and either of the 402-AX teratoma-defined antigens I and II (Edidin, 1976) is
unlikely, since these disappear on the post-implantation trophoblast of blastocyst outgrowths. Biochemical studies have, however, revealed a shift from lower
to higher molecular weight, trypsin-susceptible, carbohydrate-containing components (glycopeptides ?) on the outer surface of the embryo during transformation from the 8-cell to the blastocyst stage (Pinsker & Mintz, 1973), which
could correspond with the changes responsible for the increased detectability
of the EPC-defined antigens.
Both the ontogeny and the distribution of the EPC-defined antigens demonstrated in the present study implies that their appearance is closely associated
with the emergence of the trophectoderm as an identifiable cell population and
that, in line with evidence suggesting de novo embryonic gene activity by the
8-cell stage (Brinster, 1973; see Epstein, 1975; Muggleton-Harris & Johnson,
1976), their expression is dependent upon the embryonic genome. In addition,
the apparently trophoblast-specific nature of these determinants is indicated by
the antiserum-binding pattern in the post-implantation embryo, with heavy
labelling being confined to the trophectoderm derivatives, in contrast to the
non-labelling embryonic ectoderm and endoderm both of which are considered
154
R. F. SEARLE AND E. J. J E N K I N S O N
to be progeny of the ICM (Gardner & Papaioannou, 1975). The early differentiation of the trophectoderm thus appears to be accompanied by membrane
changes or specializations which are maintained in some of its progeny, at least
up to the early post-implantation stages, providing support for previous claims
for the existence of tissue-specific antigens on various rodent and human trophoblast populations (see Billington, 1976). The EPC contains at least two
trophoblastic cell types, the proliferating (diploid?) core cells and the outer
giant cells (Snell & Stevens, 1966; Gardner & Papaioannou, 1975). At present,
our conclusions on post-implantation trophoblast are confined to the giant cells
and further studies are required to identify the labelling characteristics of the
core cells and their progeny in the later placenta.
Although trophoblastic populations appear to be characterized by heavy
labelling, the blastomeres of the 8-cell stage and isolated ICMs show a low
level of reactivity. Perhaps the early expression of the genes involved in controlling EPC-defined products results in their appearance on the 8-cell embryo,
before some blastomeres become enclosed and subject to an 'internal' environment inhibiting further differentiation along the trophectoderm pathway,
as suggested by the 'inside-outside' theory of Tarkowski & Wroblewska
(1967). In this case the staining observed on the ICM could result from the
persistence of determinants produced at the 8-cell stage on the progeny of the
internalized blastomere(s) in which further production of trophectodermassociated determinants is suppressed. Experiments using blastomere disaggregation and recombination should allow an analysis of the effect of cell
position on the staining pattern observed with. anti-EPC antiserum.
Membrane components confined to trophoblastic cell populations, especially
those expressed at the earliest stages of development, could be considered as
potential targets for immunological fertility control procedures. Administration
of heterologous antisera raised by injection of placental homogenates is known
to result in abortion in pregnant mice and rats (Koren, Abrams & Behrman,
1968; Behrman, 1971; Beer, Billingham & Yang, 1972) but the trophoblastspecific nature of such antisera has not been conclusively demonstrated. Studies
are now in progress to determine whether anti-EPC antiserum will inhibit
early development both in vivo and in vitro and to investigate the possibility
of producing auto-immunization to tissue-specific antigens of early trophoblast
populations.
We are indebted to Dr W. D. Billington for helpful discussion and criticism of the manuscript and to Miss Vanessa Merry for technical assistance. Financial support was provided
by the Rockefeller Foundation and the Medical Research Council.
Trophoblast-defined surface antigens on mouse embryos
155
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