/ . Embryo!, exp. Morph. Vol. 69, pp. 141-149, 1982
Printed in Great Britain © Company of Biologists Limited 1982
Importance of trophoblast
genotype for survival of interspecific
murine chimaeras
By J. ROSSANT1, V. M. MAURO AND B. A. CROY
From the Department of Biological Sciences,
Brock University, St. Catharines, Ontario
SUMMARY
Interspecific chimaeras between Mus musculus and Mus caroli were made by injection of
M. musculus inner cell masses (lCMs) into M. caroli blastocysts, and by aggregation of
8-cell embryos. Both types of chimaera were transferred to M. musculus recipients: the
former did not survive to term, but viable chimaeras were produced following embryo
aggregation. Previous experiments have shown that chimaeras produced by injection of
M. caroli ICMs into M. musculus blastocysts are viable, whereas M. caroli blastocysts
cannot survive in the M. musculus uterus. These results indicate that the presence of trophoblast cells of maternal uterine genotype allows M. caroli <-> M. musculus chimaeras to survive
in the M. musculus uterus. It is suggested that M. musculus trophoblast components may
protect the M. caroli embryonic cells from maternal immune rejection.
INTRODUCTION
Successful production of live interspecific chimaeras between two murine
species, Mus musculus and Mus caroli, has recently been reported (Rossant &
Frels, 1980). This was achieved, with a high rate of normal development, by
injection of M. caroli inner cell masses (ICMs) into M. musculus blastocysts
and transfer of the resulting blastocysts into the M. musculus uterus. Cells of
the two genetically distinct species appeared to interact normally in th$se
chimaeras and the patterns and extent of mosaicism were similar to those
observed in intraspecific chimaeras (Rossant & Frels, 1980; Rossant & Chapman, in preparation). Survival of M. caroli ICM cells to term in such chimaeras
was somewhat surprising since we have observed poor survival rates of M. caroli
embryos (Frels, Rossant & Chapman, 1980), and interspecific hybrid embryos
(West et al. 1977; West, Frels & Chapman, 1978) in the M. musculus uterus.
In both these cases, most embryos were dead by day 10-15 of pregnancy: only
a very small number (c. 1 %) survived to term. Clearly M. caroli ICM c^lls
can survive in the M. musculus uterus when combined with embryonic cells of
1
Author's address: Department of Biological Sciences, Brock University, St Catharines,
Ontario, Canada, L2S 3AI.
142
J. ROSSANT, V. M. MAURO AND B. A. CROY
the uterine species type. The M. caroli cells might be afforded protection by
the M. musculus trophoblast surrounding the chimaeric ICM, or by the interactions between M. musculus and M. caroli cells in the ICM itself. To distinguish
between these possibilities we have made two further types of chimaera between
M. musculus and M. caroli and assessed their viability in the M. musculus
uterus. The first type was the 'reverse' injection chimaera, in which the ICM
was again of mixed genotype, but the trophoblast was entirely M. caroli. The
second type was the aggregation chimaera, in which both ICM and trophoblast
were expected to be of mixed genotype. The 'reverse' injection chimaeras failed
to survive to term, while the aggregation chimaeras were viable. These results
suggest that the presence of trophoblast cells of maternal uterine genotype is
necessary for survival of M. musculus«-»M. caroli interspecific chimaeras in
the M. musculus uterus.
MATERIALS AND METHODS
Recovery of embryos
Mus musculus embryos were obtained from natural matings of a closed
stock of random-bred Ha (ICR) albino, non-agouti mice, homozygous for the
b allele of the glucose phosphate isomerase (GPI) gene (Gpi-lh/Gpi-]*). Cleavage
stage 8-cell embryos were obtained by flushing the oviducts on the afternoon
of the 3rd day after mating and blastocysts were obtained by flushing the uteri
on the afternoon of the 4th day after mating. Mus caroli embryos were obtained
from a random-bred stock of the wild mice maintained at Brock University.
M. caroli is agouti in coat pigmentation and has only one allele at the
Gpi-1 locus which is indistinguishable electrophoretically from the Gpi-1&
allele of M. musculus. All embryos were obtained following hormonal treatment
with 5 i.u. pregnant mare's serum gonadotrophin (Organon) followed 48 h later
by 5 i.u. human chorionic gonadotrophin (hCG, Sigma). Eight-cell embryos
were obtained 56 h post hCG and blastocysts 76 h post hCG. PB1 medium
(Whittingham & Wales, 1969) with 10% foetal calf serum (FCS) was used
throughout for recovery and storage of embryos.
Injection chimaeras
Zona-free M. musculus blastocysts were subjected to immunosurgery (Solter
& Knowles, 1975) as described elsewhere (Rossant & Lis, 1979) and the
resulting ICMs were injected into M. caroli blastocysts by use of a Leitz
micromanipulator assembly. Injected blastocysts were allowed to recover for
1 h before transfer.
Aggregation chimaeras
Zonae were removed from 8-cell M. musculus and M. caroli embryos by
brief exposure to acid Tyrode's solution, pH 2-5, and pairs of embryos were
aggregated in small drops of a-modified MEM (Gibco) + 10% FCS under
paraffin oil (BDH) in bacteriological dishes. When embryos appeared to be
Survival of interspecific murine
143
chimaeras
Table 1. Transfer of M. caroli blastocysts injected with
M. musculus ICMs to M. musculus recipients*
Type of implantation*
Stage
analysed
11-5
14-5
16-5
Total
No.
transferred
No.
implanted
9
19
10
38
6
17
9
32
t
Live
foetus
White
foetus
3
2
0
5
1
9
6
16
1Elesorbtions
2
6
3
11
i
* Includes only pregnant females.
adhering to each other, the culture dishes were incubated at 37 °C in an
atmosphere of 5 % CO2 in air. Successful aggregates were removed from culture
24 h later and transferred to recipient females.
Transfer and analysis of chimaeras
All experimental combinations of embryos of the two species were transferred to M. musculus Gpi-lh/Gpi-lh females on the 3rd day of pseudopregnancy. Recipient females were killed at various stages of pregnancy and
any conceptuses were dissected into embryonic and extraembryonic derivatives,
and frozen before GPI analysis. Some recipients carrying aggregation chimaeras
were allowed to go to term and live young were examined for coat and eye
pigmentation and bled for GPI analysis. Electrophoretic separation of GPI
isozymes was carried out as described previously (Rossant & Us, 1979).
RESULTS
M. musculus ICM «-> M. caroli blastocysts
M. caroli blastocysts injected with M. musculus ICMs implanted in the
M. musculus uterus but only 5 live foetuses were observed out of 32 implantation
sites (Table 1). The remaining sites contained either resorbed embryos or
recently dead, white foetuses. The placentae of these white embryos were well
developed but haemorrhagic and the yolk sacs were filled with a dark yellow
fluid which presumably contained products of haemolysis. As pregnajicy
proceeded, the percentage of live foetuses declined (Table 1) from 50% at
11-5 days to 11-8 % at 14-5 days, and by 16-5 days no live foetuses were found.
All recognizable foetuses showed some eye pigmentation and GPI analysis
revealed that all were chimaeric (Table 2). There was no obvious relationship
between the proportion of M. caroli enzyme in any tissue and survival of the
foetus.
144
J. ROSSANT, V. M. MAURO AND B. A. CROY
Table 2. GPI analysis of conceptuses derived from M. caroli blastocysts
injected with M. musculus ICMs
GPI analysis*
Type of conceptus
Live
White foetusf
No.
oidgc ctiidiyoCvi
(days p.c.)
1
115
2
11-5
3
11-5
4
11-5
5
14-5
1
11-5
2
14-5
3
14-5
4
16-5
5
16-5
6
16-5
7
16-5
8
16-5
9
16-5
10
16-5
11
16-5
12
16-5
13
16 5
14
16-5
15
16-5
Embryo
Yolk sac
Placenta
3
3
3
•
3
9
9
O
3
3
3
3
3
9
3
3
3
9
3
3
3
O
3
3
9
9
9
9
9
3
3
3
3
3
0
3
(3
3
(3
3
3
9
3
3
9
3
3
3
3
3
3
3
* Black represents M. caroli clerived enzyme and white represents M. musculus derived
snzyme.
t One white foetus was not analysed.
Survival of interspecific murine chimaeras
145
Table 3. Transfer ofM. musculus *-> M. caroli aggregation chimaeras
to M. musculus recipients*
Stage analysed
9-5-15-5 days p.c.
Term or later
Total
No.
transferred
No.
recipients
No. live embryos
(% transferred)
No. chimaeric
(% live embryos)
55
35
90
7
6
13
22(400)
13 (371)
35 (38-9)
18(81-8)
7 (53-8)
25 (71-4)
* Includes only pregnant females.
M. musculus <-> M. caroli aggregates
Out of a total of 187 aggregates of 8-cell embryos from M. musculus and
M. caroli, 138 (73-8%) formed single, integrated blastocysts. This rate of
aggregation was lower than usually achieved for M. musculus <-> M. musculus
aggregates in our laboratory, reflecting the apparent non-adhesive nature of
many early 8-cell M. caroli embryos which often fell apart during zona removal.
Table 3 summarizes the results of transfer of aggregates to M. musculus females.
The rate of survival of embryos was approximately 40 % whether the embryos
were examined midgestation or after birth. This rate of live embryo production
is very similar to other experiments in our laboratory using aggregation and
culture of M. musculus embryos (Rossant & Lis, 1979, and unpublished data).
GPI analysis revealed that 25 of 35 live embryos and offspring were chimaeric
in at least some of the tissues analysed. Conceptuses studied at 9-5 days p.c.
were carefully dissected into embryonic, yolk sac and the trophectodermderived ectoplacental cone (EPC) fractions. The EPC fraction was not contaminated with ICM-derived tissues, since the portion of the EPC where the
allantois had fused was recognized and discarded. GPI analysis of these
chimaeras (Table 4) revealed that 12/16 contained M. caroli derived cells in the
ectoplacental cone. It was not possible to determine whether chimaeras dissected
later in development (Table 3) contained M. caroli cells in the trophoblast
component of the placenta since clean separation of this tissue was not readily
achieved beyond 9-5 days.
Live chimaeras produced by aggregation were identical in phenotype to
those produced by blastocyst injection (Rossant & Frels, 1980).
DISCUSSION
Interspecific chimaeras between M. musculus and M. caroli produced by
blastocyst injection were only able to survive to term in the M. musculus uterus
when the trophectoderm was M. musculus in genotype. M. caroli ICMs injected
146
J. ROSSANT, V. M. MAURO AND B. A. CROY
Table 4. GPI analysis of M. musculus *-> M. caroli aggregation
chimaeras at 9-5 days p.c.
Tissue analysed*
Embryo
no.
Embryo
O
3
O
c*
9
10
11
12
13
14
O
Yolk sac
O
Ectoplacental
cone
'9
O
o
3
o
O
O
3
O
o.
o
3
O
15
* Black represents M. caroli derived enzyme and white represents M. musculus derived
enzyme.
into M. musculus blastocysts produced viable chimaeras (Rossant & Frels,
1980), but the 'reverse' chimaeras in which M. musculus ICMs were injected
into M. caroli blastocysts did not survive beyond 16 days of pregnancy.
Trophectoderm of uterine species genotype was required for survival of the
identical chimaeric ICMs in the two types of chimaeras. We have not yet
shown whether the protection afforded the chimaeric ICM by M. musculus
Survival of interspecific murine chimaeras
147
trophectoderm can be extended to embryos in which the entire ICM is M.
caroli-derived, although the production of several viable interspecific chimaer&s
in which the M. caroli component was greater than 90 % (unpublished data)
suggests that this may be possible. Proof would require construction of
'reconstituted' blastocysts in which the TCM was M. caroli and the trophectoderm was M. musculus. Such experiments are technically difficult and the
intrinsically low viability (Gardner, Papaioannou & Barton, 1973; V. E. Papaioannou, personal communication) would make interpretation difficult.
Having demonstrated the importance of M. musculus trophectoderm for
survival of interspecific chimaeras, we expected that aggregation chimaeras,
with mosaic trophectoderm, would not survive. However, viable interspecific
chimaeras were produced by embryo aggregation. Most chimaeras analysed
at 9-5 days possessed trophoblast of mixed genotype (Table 4) and, since no
embryo loss occurred between 9-5 days and term, chimaeras of similar genetic
constitution must have been able to survive to birth. We were not, however,
able to prove that the M. caroli trophoblast cells observed at 9-5 days persisted
until term, because we could not readily separate the trophoblast layer from
the term placenta for analysis. We are currently using a new in situ marker
system, which distinguishes M. caroli and M. musculus cells in histological
sections (Rossant, Croy, Siracusa, Chapman & Clark, submitted), to examine
the constitution of the trophoblast in the placentae of viable aggregation
chimaeras. Determining whether all the trophoblast must be of uterine genotype for chimaera survival will be very important for a full understanding
of the mechanism of trophoblast protection.
There are various ways in which M. musculus trophoblast could promote
survival of interspecific chimaeras. The importance of correct trophoblast uterine interactions at implantation has been emphasized previously by work
on rat-mouse interspecific pregnancies. Rat ICMs injected into mouse blastocysts will survive beyond implantation (Gardner & Johnson, 1973), whereas
rat blastocysts transferred to the mouse fail to establish normal trophoblastuterine interactions and do not develop beyond implantation (Tarkowski, 1962;
Rossant, 1976; Tachi & Tachi, 1979). Failure of trophoblast function at
implantation cannot explain the death of M. caroli embryos and M. musculus
<-» M. caroli injection chimaeras, however, since such embryos do not die until
well beyond implantation.
Trophoblast-uterine interactions are also required for formation of the
mature chorioallantoic placenta, and abnormal placental formation between
M. musculus uterine tissue and M. caroli trophoblast might result in the death
of any enclosed foetus. However, time of death does not seem to correlate
with placental formation in the present experiments and many embryos with
M. caroli trophoblast form morphologically normal placentae (unpublished
observations). The endocrinological status of M. caroli trophoblast in such
placentae is not yet clear. In the mouse, trophoblast cells are known to secrete
148
J. ROSSANT, V. M. MAURO AND B. A. CROY
progesterone which presumably aids in embryo survival. Progesterone secretion
appears to be an intrinsic property of trophoblast cells and is not dependent
on trophoblast-uterine interactions, since trophoblast cells continue to secrete
progesterone when isolated in vitro (Sherman, Atienza, Salomon & Wudl
1977). However, M. musculus uterine tissue might interact with M. caroli
trophoblast to suppress progesterone production. Reduced local levels of
hormone might then result in embryo death as observed. We have been unable
to prolong the survival of M. caroli embryos in the M. musculus uterus by
treatment with high levels of progesterone, suggesting that this explanation is
unlikely.
A further role that has been suggested for trophoblast is that of a barrier
to immunological responses directed against foetal-borne antigens (Beer &
Billingham, 1976). When the conceptus bears antigens of a different species,
protective mechanisms may break down leading to rejection of the foetus as
a xenograft (Chang & Hancock, 1967; Dent, McGovern & Hancock, 1971).
The time and pattern of embryonic death in M. caroli and M. musculus <-> M.
caroli injection chimaeras is compatible with an immunological basis to embryo
destruction (Frels et al. 1980). We have recently obtained more direct evidence
for involvement of maternal immune responses in the death of M. caroli
embryos in M. musculus (Croy, Rossant & Clark, 1981 and manuscript submitted). Lymphocyte infiltration of the trophoblast was observed prior to any
sign of embryo death and T cells cytotoxic to M. caroli cells in vitro were
isolated from dying foetuses. If immune rejection proves to be the primary
cause of death of M. caroli embryonic cells in the M. musculus uterus, the
present experiments strongly suggest that the presence of trophoblast cells of
maternal species type can protect the xenogeneic ICM cells from immune
destruction. The M. musculus/M. caroli pregnancy system promises, therefore,
to be a useful model system for investigation of the role of trophoblast-uterine
interactions in immune protection of the foetus.
This work was supported by the Canadian Natural Sciences and Engineering Research
Council and the Banting Foundation. B.A. Croy is a Lalor Fellow. We should like to thank
Drs V. M. Chapman, and D. A. Clark for useful discussion.
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{Received 22 July 1981, revised 4 January 1982)