/ . Embryol. exp. Morph. Vol. 35, 1, pp. 59-72, 1976
59
Printed in Great Britain
The nature and distribution of
serologically detectable alloantigens on the
preimplantation mouse embryo
By AUDREY L. MUGGLETON-HARRIS 1 AND
MARTIN H. JOHNSON 2
From the Department of Anatomy, Cambridge
SUMMARY
The nature and distribution of surface alloantigens on preimplantation mouse embryos
has been examined by immunofluorescence. Non-H-2 alloantigens were detected at all
stages examined, from the 2-cell to the 4^-day blastocyst. Cleaving blastomeres, inner cell
mass cells and cells of the primary trophectoderm were all positive. In Fx embryos maternal
non-H-2 alloantigens were detectable at all stages, whereas paternal antigens first became
evident at the 6- to 8-cell stage. No convincing evidence of the presence of alloantigens
associated with the H-2 haplotype was found at any stage or on any cell type, suggesting that
if these antigens are present they are in low quantity or are masked.
INTRODUCTION
This report presents data on the alloantigenic status of the preimplantation
mouse embryo. Knowledge of the nature, quantitation and spatial distribution
of surface alloantigens on the mammalian embryo is important for several
reasons. The ontogenetic development of alloantigenic molecules and its
relationship to the presence of tumour-associated embryonic antigens may be
directly relevant to the nature of cell-to-cell interactions in developmental
processes, both within the embryo itself (Artzt et ah 1973; Artzt, Bennett &
Jacob, 1974; Edidin, Gooding & Johnson, 1975) and between embryonic and
maternal cells at implantation (Beer & Billingham, 1974). Secondly, genetically
well-defined surface determinants that can be visualized on all cells of intact
embryos are of great potential value to developmental biology (Gardner &
Johnson, 1975). Finally, an alloantigenically positive embryo might prove to be
susceptible under some conditions to immune rejection in utero, as may occur
regularly in ectopic sites (Kirby, Billington & James, 1966; Simmons & Russell,
1966; Yandeputte & Sobis, 1972).
1
Author's address for reprints: Department of Anatomy, Downing Street, Cambridge,
U.K.
2
Author's address: Department of Medical Microbiology, Stanford University, California,
U.S.A.
60
A. L. MUGGLETON-HARRIS AND M. H. JOHNSON
We have therefore undertaken a systematic analysis of mouse embryos from
2-cell to 4^-day blastocyst stages by use of an immunofluoresence technique
which permits localization of antigen on different cell populations, and with
antisera and strain combinations that permit distinction of major and minor
alloantigens in homozygous and heterozygous embryos.
MATERIALS AND EXPERIMENTAL PROCEDURES
Embryos. Six- to ten-week-old mice were induced to ovulate by i.p. injection
of 5 i.u. Pregnant Mare Serum Gonadotrophin (PMSG, Folligon, Intervet,
London), and 48 h later with 5 i.u. Human Chorionic Gonadotrophin (HCG,
Chorulon, Intervet, London) (Gates, 1971). The ovulated females were mated
and checked for vaginal plugs. One to 4^ days after ovulation the females were
killed and the embryos flushed out of the oviduct or uterus with medium PB1
plus 10 % inactivated foetal calf serum (Biggers, Whitten & Whittingham,
1971). Embryos were similarly recovered from spontaneously ovulating animals.
The zona pellucida was removed either with minimal exposure to 0-5 %
Pronase made up in a tris-citrate buffer pH 7-0 (Mintz, 1971) or non-enzymically
by use of a micromanipulator. Embryos taken at A\ days were already free of
the zona. Some early embryos were cultured in a medium defined by Whittingham (see Table 5 in Whittingham, 1971). Embryos were placed in small
amounts of medium under sterile light liquid paraffin in disposable sterile Petri
dishes and incubated at 37 °C in an atmosphere of 5 % CO2 in air (Whittingham, 1971). Some 3^- and 4^-day blastocysts were dissected with the Leitz
micromanipulator to recover inner cell mass (ICM) cells according to techniques previously published (Gardner, 1972).
Antisera. Male mice were injected I.P. with a suspension of male spleen and
lymph node cells, the cells from one animal being used to immunize four or five
recipients. In some cases the injection was preceded by a skin graft. After five
or more injections at 7- to 10-day intervals, test bleeds were made to ascertain
the toxicity of the antiserum to appropriate target lymphocytes. The blood
obtained from the final bleed was allowed to clot at 4 °C, the clear serum
pipetted off, centrifuged to remove loose cells, heat inactivated, separated into
2 ml aliquots and stored at minus 40 °C. Cytotoxic tests were made using 1 fi\
each of serum serially diluted in PB1, guinea-pig complement (1:10) and target
cells (2-9xlO 6 /mlPBl). After 30 min incubation at 37 °C, 2 fi\ of eosin
(diluted in saline) was carefully added and the slide mounted for observation.
Control counts were made using medium PB1 +10 % F.C.S., with and without
guinea-pig complement, and non-target lymphocytes in the presence of antiserum. The prepared antisera were C3H/He-mg anti-C57BL10ScSn;
C57BL10ScSn anti-C3H/He-mg; C3H/He-mg anti-C57Br-cd; C57Br-cd antiC3H/He-mg.
The following inbred strains from our laboratory were used in conjunction
Alloantigens on mouse embryos
61
with these antisera to establish the reactivity against the major and minor
alloantigens: C3H/He-mg (H-2k), C3H.SW (H-2b on a C3H background),
C57BL10ScSn (H-2b), B10.Br (H-2 k on a C57BL10ScSn background),
C57Br-cd (H-2k).
Fluorescent antibody procedures. Cytotoxic slides (Baird and Tatlock, tissue
typing slide cat. 77403/0533) were found to be satisfactory for work with the
whole embryos. Small amounts of antisera and conjugate could be used and
the numbers of wells available facilitated several washes of the embryos through
clean wells. Layering of pre-filtered light liquid paraffin on top of the slides
prevented evaporation.
Embryos were prepared and placed into the wells with a minimal amount of
medium. One /tl of specific antiserum was placed into the well using a Hamilton
precision syringe. After 35 min of incubation the embryos were carefully
pipetted through several washes of medium over a 10 min period. When
washing the embryos, enough fluid was retained in the wells to avoid compression of the embryo by the oil/water interface. One ju] of a 1/10-1/50 dilution
of the TgG fraction of a fluorescein-conjugated antiserum to mouse IgG (Miles,
Pentex; fluorchrome:protein ratio of 2:1) was then applied. After a further
incubation of 30 min the embryos were then washed carefully in medium to
remove all surplus conjugate and placed in a clean well with 2 /A of medium.
Excess paraffin was removed and a coverslip added for observation.
Incubations were carried out at 4° or 37 °C to compare the temperature
dependence of the staining pattern (Unanue, Karnovsky & Engers, 1973).
Frequently tests were made in the presence of 10~2 M sodium azide throughout
the experiment, including viewing, to prevent pinocytosis (Taylor, DufFus,
Raff & de Petris, 1971).
The following controls were undertaken: (1) embryos of the strain in which
the antiserum was made and at the same developmental stage as target embryos,
(2) use of normal mouse serum on target embryos, (3) use of medium in place
of the antiserum and (4) use of antisera absorbed previously with target lymphocyte cells, which had resulted in specific reduced toxicity for both lymphocytes
and embryos. In experiments designed to distinguish H-2 from non-H-2
antigens, the protocol for each experiment included use of age-matched
C57BL10ScSn, C3H and B10.Br embryos with both C3H anti-C57BL10 and
C57BL10 anti-C3H antisera.
Embryos were viewed on the Zeiss microscope using incident fluorescent
light source HBO 200 with excitation filter system 427902 and barrier filter
system 427903. Independent scoring by a naive observer was used wherever
possible. Photographs were taken using Kodak Recording Film 2475 with a
2 min exposure for u.v. and jj-gth sec for bright-field illumination.
62
A. L. MUGGLETON-HARRIS AND M. H. JOHNSON
RESULTS
With the use of specific antisera and combinations of strains of mice as
indicated in Table 1, the presence of alloantigens on the cell membranes of the
preimplantation embryos was demonstrated. Large numbers of individual
antisera were tested on embryos, but only a few showed good specific reactivity
and then only to titres of about 1/10. The remainder showed either non-specific
reactivity or a failure to stain; these were discarded. The antisera showed an
Table 1. Incidence of staining of embryos with differing combinations
of mice and antisera
Number and age of positive embryos
Antiserum
Strain of
embryos
C3H anti-C57BL10 C57BL10
B10.Br
C3H
C57Br
C57BL1O anti-C3H C3H
B10.Br
C3H.SW
C57BL10
C57Br anti-C3H
C3H
C57BL1O
C57Br
C3H anti-C57Br
C57Br
C3H
Antigenic
difference
1^ days
(2-4 cell)
H-2 + non-H-2
51/52
Non-H-2 only
6/7 (weak)
None
0/25
N.T.
Non-H-2 only
H-2 + non-H-2
9/9
0/11
H-2 only
N.T.
Non-H-2 only
None
0/9
Non-H-2 only
2/2 (weak)
Very few non-H-2: 0/2
0/2
None
Non-H-2 only
N.T.
None
0/4
N.T. = not tested.
2\ days
(morula)
3^- days
(blastocyst)
4^ days
(blastocyst)
48/50
7/7
0/25
4/4
9/10
0/3
3/3
0/10
2/2
0/4
0/9
3/3
0/6
51/56
10/10
0/16
5/5
21/23
0/14
3/3
0/8
3/3
0/2
0/3
3/4
0/11
36/40
10/11
0/25
3/3
34/38
0/8
4/6
0/8
7/8
N.T.
0/8
9/9
0/12
increase in non-specific reactivity with age or with repeated freezing and
thawing. For the positive antisera there was not a good correlation between
activity against embryos and the cytotoxic litre against lymphocytes, suggesting
that the target antigens on the embryo might not be the primary antigens on
lymphocytes. Convincing positive reactions were restricted to non-H-2 alloantigens. In combinations of antisera and embryos differing only at H-2 alloantigens, no clear evidence of specific staining was detected. Thus B10.Br
embryos failed to stain with C57BL10 anti-C3H antisera, whilst showing in the
same experiment fluorescence with C3H anti-C57BL10 antisera. This relative
deficiency of H-2 over non-H-2 antigens was observed at all stages and on cells
of both ICM and trophoblast. The non-H-2 antigens were present at all stages,
although at the two-cell stage (1^ days) the fluorescence appeared subjectively
to be weaker in comparison with later stages (Fig. 1). Occasionally two to four
blastomeres of the 1^- to 2^-day embryo appeared weak or negative in comparison with the other blastomeres of the same embryo. In older embryos all
cells stained (Figs. 2, 3).
Alloantigens on mouse embryos
63
Blastocysts from both !>\ and 4^ days showed a qualitatively similar result.
The trophoblast cells overlying the inner cell mass (ICM) were positive but as
seen in Fig. 4 the ICM itself appeared negative. This may have resulted from
the failure of the antiserum to penetrate the tight junctions of the trophoblast
cells. Only with advanced 4^-day embryos, in which collapse of the trophoblast
Fig. 1. Two blastomeres of a H-day C57BL10ScSn embryo treated with C3H antiC57BL10 antiserum to demonstrate the very weakfluorescenceon both membranes
of the blastomeres and the absence of fluorescence on the polar body (arrowed).
x360.
and exposure of the ICM frequently occurred, was fluorescence seen on some
inner cell mass cells. ICM cells dissected free of the trophoblast were positive
as shown in Fig. 5 and Table 2.
The fluorescence at all stages was distinct and limited to the surface membrane. There appeared to be no internal fluorescence within the blastomeres and
in the case of the 2-cell embryo (1^ days), the polar body was negative even
when the blastomeres were positive (Fig. 1). Whether the zona pellucida was
removed either with a minimal exposure to 0-5 % pronase or non-enzymically
by use of a micromanipulator, the level and distribution of antigenic markers
were similar. Use of antisera on embryos obtained from the strain in which the
antisera was raised proved negative. Occasionally, non-specific fluoresence was
observed resulting from conjugate adhering to debris trapped in the cell
64
A. L. MUGGLETON-HARRIS AND M. H. JOHNSON
junctions, but this was readily distinguishable from specific membrane
fluorescence.
Distribution of fluorescence on cells of positive embryos of all ages fell into
two categories: (a) a continuous ring of fluorescence over the membrane (see
Fig. 2), and (b) small discrete patches distributed around the perimeter of the
Fig. 2. Four blastomeres of an 8-cell embryo of C57BL10ScSn exhibiting specific
immune fluorescence after treatment with C3H anti-C57BL10 alloantisera. x 360.
blastomere membrane (see Fig. 3). The incubation of the embryos at 4 °C
favoured type (a) pattern of fluorescence which over a period of 30 min at
room temperature became type (b). This change of staining pattern occurred
regardless of whether azide was present.
The staining pattern of heterozygous embryos of different ages produced by
reciprocal matings between different strains was compared with that of homozygous parental-type embryos. Table 3 shows that at the 2-cell stage (1£ days)
Alloantigens on mouse embryos
65
there was a weak positive reaction for the products of the maternal genome but
staining for paternal alloantigens could not be scored with confidence. By the
6- to 8-cell stage Q.\ days), however, the expression of maternal alloantigens
appeared more positive, and a weak positive reaction for the paternal antigens
Fig. 3. Embryo of Fx hybrid C3H?x C57BL10ScSn 61 after treatment with C3H
anti-C57BL10ScSn antisera. x 360.
was unequivocally detectable. By 8-12 cells the paternal antigens were judged
to be of similar strength to maternal antigens, although occasionally two or
three of the blastomeres were weak or negative for both antigens. The 1\- to
4^-day embryos showed similar levels of positive fluorescence reactions for
5
EMB
35
66
A. L. M U G G L E T O N - H A R R I S AND M. H. JOHNSON
4A
Fig. 4. (A) 4i-day C57Br-cd blastocyst treated with C3H anti-C57Br alloantisera to
demonstrate positive membranefluorescenceof the trophoblast cells, but no visible
fluorescence on the cells of the inner cell mass, x 360. (B) Phase contrast of the
same 4^-day blastocyst. x 360.
Alloantigens on mouse embryos
67
5B
Fig. 5. (A) Cells from inner cell mass of C57Br 4^-day blastocyst dissected free of
trophoblast demonstrating that the majority of these cells show positive fluorescence
on their membranes, x 360. (B) Phase contrast of the same inner cell mass cells.
x360.
5-2
68
A. L. M U G G L E T O N - H A R R I S AND M. H. JOHNSON
both paternal and maternal antigens. Identical results were obtained using
embryos cultured from the 2-cell stage (1^ days) to late morula, precluding any
possibility that paternal antigens might be picked up from residual components
of semen on entry of the eggs into the uterus. Selection of samples of cultured
embryos at each successive division permitted more precise estimation of the
appearance of paternal antigens, which were detected with confidence as early
as the 6-cell stage. Fig. 3 shows a morula Fx hybrid with fluorescing stain
patching on the blastomere membranes.
Table 2. Staining of dissected inner cell mass cells in specific antisera
(All incubations at 0 °C and in azide.)
Age of donor
blastocyst
Antiserum
C3H anti-C57BL10
C57BL10 anti-C3H
Strain of
ICM
Antigenic
difference
C3H
C57B10
BlO.Br
C3H
C57B10
BlO.Br
C3H.SW
None
H-2 + non-H-2
Non-H-2 only
H-2 + non-H-2
None
H-2 only
Non-H-2 only
N.T. = not tested.
A\ days
days
0/3
5/5 (weak)
N.T.
1/1 (weak)
0/2
0/1
N.T.
0/2
6/7
4/4
3/3 (weak)
1 /4 (weak)
0/3
4/5 (weak)
Table 3. Detection of maternal and paternal antigenic expression in Fx embryos
Antiserum
C3H anti-C57BL10
Fi hybrid
combinations
?
$
C3H x C3H
C3HxC57BL10
C57BL10xC57BL10
C57BL10xC3H
1^ days
2{- days
2 days
(2-4 cells) (6-8 cells) (8 cells+ )
0/10
0/10
9/9 (weak)
8/8 (very
weak)
0/6
8/8 (weak)
8/8
6/6 (weak)
0/5
4/4
5/5
5/5
3^ days
(blastocyst)
4£ days
(blastocyst)
0/6
2/2
4/4
6/6
0/4
4/4
4/4
7/7
DISCUSSION
Previous experimental attempts to identify alloantigens on preimplantation
embryos have approached the problem by use of both transplantation and
serological techniques. In most studies in which embryos appeared to possess
alloantigens, differences at both major and minor histocompatibility loci were
involved (Kirby et al. 1966; Simmons & Russell, 1966; Olds, 1968; Vandeputte
& Sobis, 1972). However, when combinations of antisera and embryos mono-
Alloantigens on mouse embryos
69
specific for products of the H-2 haplotype have been tested (Heyner, Brinster &
Palm, 1969; Palm, Heyner & Brinster, 1971; Gardner, Johnson & Edwards,
1973), embryos from 2-cell to blastocyst stages have been found to be negative.
Similarly, grafting between strains congenic for H-2 has not resulted in blastocyst rejection despite a potent state of allograft immunity (Searle et al. 1974).
In contrast, use of antisera directed solely against non-H-2 specificities (Palm
et al. 1971) or of graft rejection across multiple non-H-2 differences (Searle et al.
1974; Hetherington & Humber, 1975) has yielded evidence favouring the
presence of those alloantigens on mouse embryos.
In this study of over 700 embryos we have confirmed the difficulty of detecting
antigenic products associated with the H-2 locus on preimplantation embryos
(Heyner, 1973; Patthey & Edidin, 1973) and demonstrated that in contrast
non-H-2 antigens are detectable on all cells during cleavage stages and the
primary differentiation into ICM and trophoblast. The appearance of the
antigens on trophoblast cells is evidently transient since use of the same antisera
on blastocyst outgrowths in culture resulted in staining of the presumed inner
cell mass cells but failure to stain trophoblastic giant cells (unpublished data).
Searle & Billington (personal communication), using peroxidase-labelled
alloantibodies, have likewise detected alloantigens on the trophoblast cells of
blastocysts but not on trophoblast outgrowths. Other reports confirm the
suggestion that trophoblast maturation may be accompanied by loss of detectable antigenicity, which could result from failure of antigen expression or
possibly from masking (Heyner, 1973).
In our study the nature of the non-H-2 antigens detected was not defined but
several may have been present. We cannot therefore claim that any one antigen
is present at all preimplantation stages and on all cell types. Neither has the
changing expression of alloantigenic determinants yet been quantified, although it appears by subjective assessment to increase during cleavage, blastomeres staining weakly positive or even occasionally negative until the 6- to
8-cell stage and thereafter increasing in intensity. Heterogeneity of blastomeres
in respect of other markers such as enzymes has previously been reported for
cleaving embryos (Dickmann & Dey, 1974). Differentiation in early blastomeres may depend on the number of cleavage divisions undergone rather than
the age of the embryo (Izquierdo & Ortiz, personal communication) and, since
cell division is asynchronous in the 5- to 16-cell embryo (Barlow, Owen &
Graham, 1972), differential changes of antigen and enzyme activity over this
period are not surprising. Changes in alloantigen availability during the cell
cycle could also lead to variation between blastomeres. The general increase in intensity of reaction as cleavage proceeds appears to occur on embryos developing
both in vivo and in vitro, suggesting an independence from maternal regulation.
The increase could either be under the control of newly synthesized or activated
embryonic template, or due to progressive unmasking or reorganisation of
presynthesized antigen.
70
A. L. MUGGLETON-HARRIS AND M. H. JOHNSON
A de novo synthesis of antigens from embryonic template seems the more
probable explanation in view of the first appearance over the same early
cleavage period of paternal alloantigens in heterozygous embryos. Although
this observation could be explained by activation of mRNA carried over by the
fertilizing spermatozoa, several lines of evidence suggest that de novo activity
by the embryonic genome may be responsible. Thus biochemical studies on
mouse embryos have provided both indirect and direct evidence for essential
gene transcription very soon after fertilization (Knowland & Graham, 1972;
Van Blerkom & Brockway, 1975). At least some of this synthetic activity seems
to involve activation of the embryonic genome. Thus, by the morula/blastocyst
stage the activities of X-linked enzymes correspond to that of the embryonic
rather than maternal chromosomal complement (Epstein, 1972) and traces of
the paternal variant of glucose phosphate isomerase may be observed as early
as the 8-cell stage (Brinster, 1973). The present use of alloantigens as a marker
confirms these previous indications of early paternal gene expression but with
the advantage that individual embryos can be examined without destruction of
embryonic organization and cell contact. The technique also permits the transfer
of embryos after examination and typing to pseudo-pregnant recipients with a
successful pregnancy rate to term of approximately 25 %, a possibility that
might permit selection of embryos (unpublished data).
The failure to detect antigens associated with the H-2 haplotype, despite
evidence to indicate active expression of non-H-2 alloantigens, may merely
reflect the insensitivity of our assay at detecting low levels of H-2. However, a
variety of techniques have now failed to provide convincing evidence for H-2
antigens on early embryos. This relative deficiency of H-2 antigens presumably
has relevance to the general biological roles of the determinants (Johnson, 1975).
Antigens controlled by the H-2 haplotype seem to be involved entirely in
immunological phenomena and first appear in embryos shortly before the time
when cells immunocompetent to induce GVH reactions are first detectable
(Hofman & Globerson, 1973). The apparent deficiency of H-2 antigens on
cells of the peri-implantation embryo is evidently reciprocally related to high
levels of an antigen carried on an undifTerentiated teratoma. Undifferentiated
teratoma cells show a similar deficiency of H-2 determinants, but since they
incite an active rejection response when grafted between strains differing at
multiple H-loci, they presumably express, like the early embryo, non-H-2
transplantation antigens (Edidin et al. 1975).
Athough the precise identity of the alloantigens detected in our study is not
known, they offer us a potentially useful marker for analysis of intercellular
relationships in early embryos. Already considerable information about cell
movement, commitment and interaction during embryogenesis has accrued
from the use of antigenic markers on interspecific chimaeras between rat and
mouse (Gardner & Johnson, 1975). The same sort of analysis applied to interstrain chimaeras making use of alloantigenic markers could substantiate these
Alloantigens on mouse embryos
71
observations in an experimental situation which was biologically more natural.
Finally, the alloantigenic determinants whilst being a useful tool could also
pose a potential hazard to the survival of the embryo. Since Fx embryos express
paternal antigens, and since previous experiments have shown that foreign
alloantigens constitute a suitable target for mediating rejection of ectopic
embryonic grafts (Searle et al. 1974), the embryos in utero might also be vulnerable to immunological attack under some conditions.
We wish to thank Virginia Papaioannou and Sheila Barton for their help and advice, and
Janis Ingram-Johnson for patient preparation of the manuscript. Supported by a M.R.C.
project grant to M.H.J.
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{Received 13 October 1975)