/. Embryol. exp. Morph. Vol. 33, 3, pp. 731-744, 1975
Printed in Great Britain
731
Development of mouse-bank vole interspecific
chimaeric embryos
By EWA T. MYSTKOWSKA 1
From the Laboratory of Experimental Embryology, Institute of Obstetrics
and Gynaecology, Warsaw, and the Department of Embryology,
Zoological Institute, University of Warsaw
SUMMARY
One bank vole {Clethrionomys glareolus) embryo and two mouse embryos were combined
at the 8- to 16-blastomere stage and cultured in vitro for 33-47 h. In 66% of cases single
regular blastocysts were formed. The chimaeric composition of blastocysts was confirmed
karyologically. Out of the 222 blastocysts transplanted to 49 pseudopregnant mouse recipients,
a total of 52 implantations were found in 20 recipients. Among the 52 implantations, 14
contained embryos and the remaining were resorptions. The majority of embryos were
abnormal and fell into two categories: (1) groups of cells surrounded by Reichert's membrane
and lying freely in a cavity filled with giant trophoblastic cells, (2) small and retarded eggcylinders usually composed of endoderm and ectoderm only, and containing a proamniotic
cavity. The ectoplacental cone of these embryos was poorly developed or lacking altogether.
Two normal-looking embryos were recovered on the 9th and 10th day (4-somite and ca.
12-somite stage). Chimaerism of the younger embryo was confirmed karyologically. No
evidence of chimaerism was available in the case of older embryo which was examined histologically. Thirteen implantations examined between 11th and 17th day contained only
resorptions. It is suggested that the main cause of the heavy mortality of chimaeric embryos
is the profound difference in the course of embryogenesis of these two species immediately
following implantation.
INTRODUCTION
Chimaeras are defined as individuals which contain cells derived from two
or more zygotes. In the mouse, chimaeras can be routinely produced by combining cleaving embryos at the 8- to 16-cell stage (Tarkowski, 1961; Mintz,
1962). Recently reports have been published describing the combination of
mouse and rat cleaving eggs to form a single blastocyst (Mulnard, 1973; Stern,
1973; Zeilmaker, 1973). Gardner & Johnson (1973) obtained chimaeric blastocysts by a different technique. They transplanted the whole inner cell mass of
the rat blastocyst into the mouse blastocyst and succeeded in obtaining development of such interspecific chimaeric embryos up to the 10th day of pregnancy.
In the present study interspecific chimaeric embryos were produced by combining eggs of the bank vole {Clethrionomys glareolus) and of the mouse. These
1
Author's address: Laboratory of Experimental Embryology, Institute of Obstetrics and
Gynaecology, Medical Academy, 00-315 Warsaw, Karowa 2, Poland.
46
EM B 33
732
E. T. MYSTKOWSKA
two species are taxonomically distant as they belong to two different families,
and they differ in the course of the early post-implantation development
(Ozdzenski & Mystkowska, in preparation). It seemed interesting to find out
whether the cleaving embryos of these two taxonomically remote species could
integrate to form a single blastocyst and, if so, how far such chimaeric embryos
could develop beyond implantation.
MATERIAL AND METHODS
Swiss albino mouse and bank vole females were induced to ovulate with 5 i.u.
PMSG ('Gestyl', Organon) and 5 i.u. HCG ('Biogonadyl', Biomed) given at
an interval of 35^48 h. Both mouse and vole eggs were collected at the 8- to
16-cell stage. The procedure for combining the eggs and culturing them to the
blastocyst stage was essentially similar to that used for obtaining mouse chimaeras and was described in our earlier papers (Tarkowski, 1961; Mystkowska &
Tarkowski, 1968; Mystkowska, 1974). The main difference was that three rather
than two eggs were combined, namely one vole egg was placed centrally between
two mouse eggs. It was hoped that such an arrangement would increase the
chance of the formation of trophoblast from the mouse blastomeres which, in
turn, would facilitate implantation of the embryo in the mouse recipient. The
embryos were inspected 1-3 h after the start of culture, to make sure that they
had remained stuck together. The embryos were cultured in a slightly modified
Mulnard's medium (personal communication, the full composition is described
in a paper by Mystkowska, 1974). They were kept in culture for 33-34 h or
43-47 h. Chimaeric blastocysts were either examined in air-dried preparations
(Tarkowski, 1966) or transplanted to the uterus of pseudopregnant Swiss albino
females mated to vasectomized males. Transplantations were performed on the
evening of the third day (20.00-22.00) or on the morning of the fourth day
(09.00 to noon), counting the day on which the vaginal plug was found as the
FIGURES
1-13
Figs. 2, 5, 7, 9 and 4, 8, 10 represent successive stages of development of the same
triplets.
Figs. 1-2. Bank vole embryo (in the middle) joined with two mouse embryos, x 200.
Figs. 3-5. Chimaeric morulae after 21 h of culture, x 200.
Figs. 6-8. Chimaeric blastocysts after 44 h of culture, x 200.
Figs. 9-10. Blastocysts shown in Figs. 7 and 8 after additional 5 h of culture, x 200.
Fig. 11. Fragment of air-dried preparation made from chimaeric embryo no. 7,
showing three mouse metaphase plates and one vole metaphase plate. Arrows
indicate plates shown in enlargement in Figs. 12 and 13: long arrow - mouse metaphase plate, short arrow - vole plate, x 100.
Fig. 12. Bank vole metaphase plate, x 1000.
Fig. 13. Mouse metaphase plate, x 1000.
Mouse-bank vole chimaeric embryos
733
10
»
A
13
46-2
734
E. T. MYSTKOWSKA
first day. Recipients were sacrificed from 6th to 18th day of pregnancy. Uteri
with implantations (6th to 10th day) were fixed in a solution consisting of 14
parts of 96 % ethyl alcohol, 5 parts of formalin and 1 part of glacial acetic acid.
Sections were cut at 6 /tm and stained with haematoxylin and eosin. From one
embryo recovered on the 9th day karyological preparations were made by the
method of Evans, Burtenshaw & Ford (1972). Implantations found in females
killed between 11th and 18th day were torn open and inspected under dissecting
microscope.
RESULTS
1. Preimplantation development in vitro
Altogether 784 mouse and 392 vole eggs were used. From 392 triplets 258
single blastocysts (65-8%) were obtained. In 55 cases (14-0%) three or two
separate blastocysts developed while in 79 cases (20-1 %) one, two or all three
eggs underwent degeneration. These results compare well with control experiments in which three mouse eggs were combined together. Out of 95 triplets
79 single blastocysts (79%) were obtained. In two cases (2%) two separate
blastocysts developed, and in 18 cases (19%) one, two or all three eggs degenerated. Two females into which 11 blastocysts originating from three mouse
eggs were transplanted gave birth to seven young.
Vole eggs differ in appearance from mouse eggs, in that they are smaller and
darker due to abundant granules present in the cytoplasm (Figs. 1, 2). The
difference in the degree of granulation makes it possible to see in chimaeric
morulae the boundaries between the contributing embryos even after 21-24 h
of culture. At this moment in the compact and usually elongated morula, the
darker vole blastomeres are grouped centrally and the lighter mouse blastomeres are on the sides (Fig. 5). As cell divisions progress and the morula rounds
up the borders between the three components become obliterated. In some
cases, however, already after 24 h of culture it is no longer possible to distinguish
the outlines of the three eggs (Figs. 3, 4). The blastocoel appears after about
44 h of culture in the form of one (Fig. 8) or two (Figs. 6, 7) cavities. In the
latter case the two cavities merge together after a few hours and a typical blastocyst is formed (Figs. 9, 10). Chimaeric blastocysts derived from three eggs consisted on the average of 87-8 cells (including 10-1 metaphase plates; mean from
seven blastocysts). In air-dried preparations both mouse and vole metaphase
plates were found.
Only regular blastocysts in which all blastomeres had been incorporated were
selected for transplantation.
2. Post-implantation development
A total of 222 blastocysts were transplanted to 49 recipients. Altogether 52
implantations were found in 20 recipients (Table 1).
Mouse-bank vole chimaeric embryos
735
Table 1. Post-implantation development of chimaeric embryos
Day of
dissection
of recipient
6
7
8
9
10
11
12
14
15
17
18
Total
No. of
recipients
No. of
blastocysts
transplanted
3
3
4
6
17
3
5
4
1
1
2
49
9
8
19
27
96
14
21
12
3
4
9
222
No. of
implantations
(no. of
No. of recitransplanted
pients with
blastocysts in
implantations parentheses)
1
2
2
4
6
1
—
2
1
1
—
20
3(3)
6(6)
5(9)
8 (14)
17 (30)
3(5)
—
6(7)
1(3)
3(4)
—
52 (81)
No. of
embryos
1
3
2
5
3
—
—
—
—
—
—
14
(a) Morphology of embryos recovered between the 6th and 10th
day of development
Sixth day of development
Embryo no. 1 (Fig. 14). The egg-cylinder is elongated and consists of an outer
layer of flattened endodermal cells, and an inner layer of round or oval ectodermal cells. The ectoderm is subdivided into embryonic and extra-embryonic
parts. The ectoplacental cone is not visible.
Seventh day of development
Embryo no. 2 (Fig. 15). The embryo is smaller than normal mouse and vole
embryos of the same age and much less advanced in development. It is normally
implanted and has a well-developed ectoplacental cone and a Reichert's membrane. Ectoderm and endoderm can be distinguished but there is no proamniotic
cavity.
Embryo no. 4 (Figs. 16, 17). The egg-cylinder is composed of endoderm and
ectoderm which is subdivided into embryonic and extra-embryonic parts. The
upper part of the cylinder resembles that of a bank vole rather than a mouse
cylinder, in that instead of the ectoplacental cone there is a groove opening
into the lumen of the uterus (Fig. 16) (Ozdzeriski & Mystkowska, in preparation).
The embryo is surrounded by a Reichert's membrane and a layer of trophoblastic giant cells.
Embryo no. 3 (Fig. 18). This embryo was collected from the same recipient
as embryo no. 2. It is represented by a group of embryonic cells situated in the
middle of a spongy structure of decidual origin filled with blood.
E. T. MYSTKOWSKA
tik;{
23
Mouse-bank vole chimaeric embryos
111
Eighth day of development
Embryo no. 5 (Fig. 19). The embryo has reached the stage corresponding to
a 7-day mouse egg-cylinder. The ectoderm is subdivided into embryonic and
extra-embryonic parts and the proamniotic cavity is present. There is no ectoplacental cone. The embryo is surrounded by spongy decidual tissues containing giant trophoblastic cells in its meshes. The band of decidual tissue
around the embryo is degenerating and the trophoblastic layer together with
the Reichert's membrane is embedded in blood.
Embryo no. 6 (Fig. 20). This dead embryo was collected from the same
recipient as the previous one. It consists of a group of cells embedded in the
Reichert's membrane-like matrix and lies freely in the mesometrial part of the
decidual crypt. The crypt is surrounded by giant trophoblastic cells and is
filled with blood.
Ninth day of development
Embryo no. 7. This embryo was dissected from the uterus and examined
karyologically. It had four pairs of somites and in its appearance and degree of
FIGURES
14-23
Chimaeric embryos, 6th-9th day of development (mesometrial pole oriented
upwards).
Fig. 14. Embryo no. 1, 6th day of development. Ectoderm already subdivided into
embryonic and extra-embryonic part, x 270.
Fig. 15. Embryo no. 2, 7th day of development. Embryonic and extra-embryonic
parts of the cylinder and the ectoplacental cone are visible, x 300.
Figs. 16,17. Two slightly oblique sections through embryo no. 4, 7th day of development. Left section shows groove in the upper part of the cylinder, characteristic for
the vole embryo. Right sectfon shows subdivision of ectoderm into embryonic and
extra-embryonic parts, x 280.
Fig. 18. Embryo no. 3, 7th day of development. Group of embryonic cells. x430.
Fig. 19. Embryo no. 5, 8th day of development. Slightly oblique section showing
embryonic and extra-embryonic parts. This egg-cylinder is deprived of an ectoplacental cone, x 230.
Fig. 20. Embryo no. 6, 8th day of development. Group of embryonic cells embedded
in Reichert's membrane lie in a cavity occupied by giant trophoblastic cells and
extravasated blood, x 80.
Fig. 21. Two embryos, nos. 8 and 9, 9th day of development, implanted one above
the other. The embryos are surrounded by a network formed by loose decidual
tissue and giant trophoblastic cells, x 80.
Fig. 22. Larger embryo (no. 8) from Fig. 21 under higher magnification. Beginning
of mesoderm formation, x 180.
Fig. 23. Embryo no. 11, 9th day of development. Degenerating two-layeied eggcylinder. Loose network of decidual cells and giant trophoblastic cells surrounds
the cylinder, x 110.
738
E. T. MYSTKOWSKA
Mouse-bank vole chimaeric embryos
739
development it resembled a 9-day-old mouse embryo, with the exception that
the ectoplacental cone was rather small. Its dimensions within the foetal membranes were 2x1-5 mm. Eighty-two mitotic plates were examined, 70 of which
had a mouse karyotype and 12 a bank vole karyotype (56 chromosomes including
a pair of small metacentric chromosomes) (Figs. 11-12, 13).
Embryos nos. 8 and 9 (Figs. 21, 22). These two embryos were found in one
implantation crypt, implanted close together. The embryos lie in a network
composed of decidual cells and giant trophoblastic cells. Each of them is surrounded by its own Reichert's membrane lined outside with trophoblast and
inside with distal endoderm.
The embryo lying in the antimesometrial part of the crypt is larger and more
advanced in development (Fig. 22). Both extra-embryonic endoderm (highly
vacuolated cells) and embryonic endoderm (flat cells) are present. The major
part of the egg-cylinder is covered with extra-embryonic endoderm which is
folded in many places. The ectoderm is multilayered and subdivided into
embryonic and extra-embryonic parts. Mesoderm formation has just begun.
The proamniotic cavity is very large. The ectoplacental cone is absent and the
embryo is not directly attached to the uterus.
The second embryo is less advanced in development. Like the first one it
lacks an ectoplacental cone and is surrounded by a network of giant trophoblastic cells. The embryo consists of an egg-cylinder attached to a cellular mass
of unknown character.
Embryo no. 11 (Fig. 23). This embryo was a degenerating egg-cylinder surrounded by Reichert's membrane. The surrounding endometrium, which contained disseminated giant trophoblastic cells, shows signs of advanced degeneration. The embryo has no contact either with Reichert's membrane or the
uterine tissues.
Embryo no. 10 (Fig. 24). The embryo was recovered from the same recipient
F I G U R E S 24-28
Chimaeric embryos, 9th-10th day of development (mesometrial pole oriented
upwards).
Fig. 24. Embryo no. 10, 9th day of development. Group of cells of embryonic origin
surrounded by Reichert's membrane lying at the bottom of a large cavity filled with
blood. Giant trophoblastic cells occupy peripheries of the cavity, x 130.
Fig. 25. Normal-looking embryo no. 12, 10th day of development, x 16.
Fig. 26. Abnormal embryo no. 13, 10th day of development. See text for detailed
description, x 110.
Fig. 27. Dead embryo no. 14, 10th day of development. Mass of degenerated cells
surrounded by Reichert's membrane. Decidual and giant trophoblastic cells have
also degenerated, x 100.
Fig. 28. Giant trophoblastic cell characteristic for the bank vole, lying at the border
of endometrium and myometrium of the mouse uterus. This giant cell was found in
the implantation swelling containing embryo no. 14. x 130.
740
E. T. MYSTKOWSKA
as embryos nos. 8 and 9. It is represented by a group of cells embedded in a
matrix corresponding in character to Reichert's membrane. The abortive eggcylinder lies in the antimesometrial part of a large crypt formed by loose decidua
and giant trophoblastic cells and filled with blood. It resembles the embryo
no. 6 described above.
Tenth day of development
Embryo no. 12 (Fig. 25). This was a healthy embryo at the stage of ca. 12
somites. The embryo corresponds to a normal mouse embryo of the same stage.
Embryo no. 13 (Fig. 26). This was a litter-mate of embryo no. 12. It consists
of a small compact body with a fissure in the middle and a loose mass of cells
partly encompassing the compact body. Signs of degeneration are already visible.
Adjacent to this structure is a mass of healthy cells, with large heavily stained
nuclei; these cells are probably in the process of transformation into trophoblastic giant cells. All these structures are surrounded by a network of giant
trophoblastic cells and are not directly connected with the decidua.
Embryo no. 14 (Fig. 27). In this case there was a mass of degenerated cells
surrounded by Reichert's membrane. Both the giant trophoblastic cells and
those of the decidua exhibit signs of advanced degeneration. Deep in the endometrium and close to myometrium, eight healthy looking giant cells were found
(Fig. 28). By their size and location these cells correspond to giant cells characteristic of the bank vole (Brambell & Rowlands, 1936). Their dimensions ranged
from 50 x 38 to 127 x 108 jum (mean 78 x 59 /mi). In the second implantation
swelling from the same horn, the only traces of an embryo that could be detected
were two giant trophoblastic cells (dimensions 85 x 46 and 96 x 77 /mi), again
embedded deeply in the endometrium.
(b) Fate of chimaeric embryos in the second half of pregnancy
In order to find out how far chimaeric embryos can develop, seven females
were chosen in which pregnancy was recognized on the 9th day on the basis of
the presence of abundant mucus in vaginal smears. The females were kept
alive until the appearance of cornified cells in the smears, which was taken as
a sign of termination of pregnancy and which occurred in all seven animals
between the 11th and 18th day (Table 1). Altogether 11 resorptions were collected and examined under a dissecting microscope. Five resorptions - one
recovered on the 1 lth, one on the 14th day and three recovered on the 17th day contained large amounts of embryonic tissue, which suggested that the embryos
must have died in the second half of pregnancy.
DISCUSSION
The present experiments show that cleaving eggs of the mouse and of the
bank vole can integrate to form single and regular blastocysts. Inspection
Mouse-bank vole chimaeric embryos
741
during culture and karyological examination of a number of blastocysts provided evidence that cells of both species were present at this stage and were
undergoing mitosis. However, it is not known whether the initial ratio of cells
(2:1 in favour of the mouse) was maintained at the blastocyst stage, and what
the spatial distribution of these two kinds of cells was within chimaeric blastocysts. One mouse embryo was placed on each side of a bank vole embryo in
order to increase the chances of trophoblast formation from the mouse cells
and thus to facilitate implantation in mouse recipients. The experiment was
designed in this way in view of the work of Tarkowski, who transplanted rat
blastocysts to the mouse and vice versa (1962), and field vole (Microtus agrestis)
blastocysts to the bank vole (personal communication), and who found that
the blastocysts of different species, although they produced the decidual reaction,
could not establish contact with the uterine mucosa and died shortly after
implantation.
The implanted chimaeric embryos may be classified into the following groups:
(1) Embryos consisting of a mass of cells lying in a cavity filled with giant
trophoblastic cells and showing no direct contact with the uterus (embryos
nos. 3, 6, 10, 14).
(2) More or less normally developed egg-cylinders. The ectoderm, endodern
and the proamniotic cavity (in the case of 7-day-old or older embryos) are
always distinguishable. Formation of mesoderm was observed in one embryo.
Embryos either lack the ectoplacental cone or else the latter is poorly developed
(embryos nos. 1, 2, 4, 5, 8, 9, 11, 13).
(3) Normally organized embryos (embryos nos. 7, 12).
The embryos belonging to the second group, which is most numerous, present
a wide scale of variability, from egg-cylinders, which were almost normal but
with smaller ectoplacental cones than mouse embryos, up to irregular and small
two-layered egg-cylinders showing no direct contact with the endometrium.
Frequently one horn contained embryos differing considerably in structure and
stage of development (for example embryos nos. 2 and 3, 5 and 6, 12 and 13).
These observations suggest that the distribution of both components in chimaeric
blastocysts as well as the contribution of vole cells to the trophoblast varied
from case to case and that this factor was mainly responsible for the different
behaviour of embryos in the post-implantation period.
The coexistence of vole and mouse cells in chimaeric blastocysts may lead to
serious developmental disturbances as early as the time of implantation. Transformation of the blastocyst into an egg-cylinder takes a different course in the
vole and in the mouse (Ozdzeriski & Mystkowska, in preparation). In the mouse
the ectoplacental cone is large and well developed right from the beginning,
whereas in the vole it is originally lacking and the lumen of the egg-cylinder
opens into the uterine lumen. A solid ectoplacental cone arises much later and
it is smaller than that of the mouse. Only in a few cases does this region of
chimaeric embryos take a form characteristic of the mouse (nos. 2, 7, 12) or the
742
E. T. MYSTKOWSKA
vole (no. 4). In most cases the embryos lie loosely in the uterus without developing direct connexion with the decidual tissue. Under these circumstances
early death of the embryo and, consequently, degeneration of the surrounding
decidual tissue is inevitable. Disturbances due to the difference in the course of
development of the two species may be expected during the whole embryonic
life of an interspecific chimaera. However, the later the stage at which such
incompatabilities occur, the smaller is the chance that they would result in
immediate embryo death. In the case of the vole and the mouse profound differences occur at the first stage of post-implantation development and this was
probably the main cause of heavy mortality of chimaeric embryos.
Although the spatial arrangement of the bank vole and mouse eggs at the
moment of fusion was always the same, the distribution of vole and mouse cells
in chimaeric blastocysts varied. Because of the position of the vole egg between
two mouse eggs, most of the vole cells in the majority of blastocysts must have
contributed to the inner cell mass. However, in some blastocysts part of the vole
cells must have remained in the trophoblast as indicated by the presence of vole
giant trophoblastic cells in the mouse uterus. Development of the mesometrial
part of the chimaeric egg-cylinder in a way characteristic of the vole rather
than of the mouse may be interpreted either as indicating the presence of vole
cells in the trophoblast covering the inner cell mass, or as a result of an inductive
influence of the vole inner cell mass on the mouse trophoblast. According to
Gardner & Johnson (1972), formation of the ectoplacental cone in the mouse
depends on the influence of the inner cell mass.
The technique of producing interspecific chimaeras employed by Gardner &
Johnson (1973), i.e. inoculation of the inner cell mass from the blastocyst of
one species (rat) into the blastocoel of a blastocyst of another species (mouse)
is superior to the technique used in this study in that the inner mass becomes
chimaeric, while the trophoblast remains homogenous. Rat-mouse chimaera
embryos develop normally up to the 10th day at least. It should, however, be
borne in mind that embryonic development of the mouse and rat is much more
similar than that of the mouse and bank vole and hence developmental disturbances are much less likely to occur in the former than in the latter combination.
Two normal-looking embryos found on the 9th and 10th day of development
reached a rather advanced stage (four-somite embryo and ca. 12-somite embryo).
Chimaerism of the 9th day embryo was confirmed karyologically. It had a small
ectoplacental cone which in the course of further development might have caused
disturbances in the formation of the placenta. The 10-day-old embryo was quite
normal. Since it was only histologically examined no proof of its chimaerism is
available. The conclusion that it consisted solely of mouse cells would require
the assumption that all vole cells were eliminated in the post-implantation
period. Among mice developed from two fused embryos there are always individuals in which the presence of only one component can be detected (Myst-
Mouse-bank vole chimaeric embryos
743
kowska & Tarkowski, 1968; Mintz, 1969; Mullen & Whitten, 1971). At least
in some of these cases the second component might have been included as a
whole into the trophoblast or extra-embryonic membranes. As explained above
(p. 741), in the case of mouse-bank vole chimaeras, the contribution of
all or the majority of bank vole cells to the trophoblast is likely to cause disorders
in implantation and early death of the embryos. Such a situation could not
therefore have existed in the embryo in question. One cannot of course exclude
the possibility that one component is overgrown by the other and either totally
eliminated or reduced to a negligible level. The author has studied a mouse
chimaera, in the bone marrow of which the ratio of metaphase plates of the two
components was 99:1 and in the cornea 20:2 (Mystkowska, unpublished results).
The 10-day-old embryo referred to above (no. 12) may represent an example of
such an 'apparent' chimaera; that is, a chimaera in which one component is
represented in a negligible and developmentally insignificant amount.
It is possible that in the case of combining eggs of taxonomically so distant
species as mouse and vole, only such chimaeric embryos in which the cell line
alien to the recipient is limited to the embryo itself and is represented in small
amount only have a chance of longer development.
The author wishes to express her thanks to Professor A. K. Tarkowski for his advice and
guidance in the course of the present work. She is also indebted to Dr W. Ozdzenski for
helpful discussions and for preparing photographic documentation.
Bank voles used in the present study were obtained from the Mammals Research Institute
in Biatowieza, Poland. The author wishes to express her thanks to Professor Z. Pucek and
mgr A. Buchalczyk for making the animals available to her.
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{Received 15 August 1974, revised 14 October 1974)